Bio


Using and Understanding Cell-Free Biology

Swartz Lab General Research Focus:

The current and projected research in the Swartz lab balances basic research in microbial metabolism, protein expression, and protein folding with a strong emphasis on compelling applications. The power and versatility of cell-free methods coupled with careful evaluation and engineering of these new systems enables a whole new range of applications and scientific investigation. Fundamental research on: the mechanisms and kinetics of ribosomal function, fundamental bioenergetics, basic mechanisms of protein folding, functional genomics, and metabolic pathway analysis is motivated by a variety of near- and medium term applications spanning medicine, energy, and environmental needs.

Swartz Lab Application Focus:

In the medical area , current research addresses the need for patient-specific vaccines to treat cancer. Particularly for lymphomas, there is a strong need to be able to make a new cancer vaccine for each patient. Current technologies are not practical for this demanding task, but cell-free approaches are rapid and inexpensive. We have already demonstrated feasibility in mouse tumor challenge studies and are now expanding the range of applications and working to improve the relevant technologies. Experience with these vaccines has also suggested a new and exciting format for making inexpensive and very potent vaccines for general use.

To address pressing needs for a new and cleaner energy source, we are working towards an organism that can efficiently capture solar energy and convert it into hydrogen. The first task is to develop an oxygen tolerant hydrogenase using cell-free technology to express libraries of mutated enzymes that can be rapidly screened for improved function. Even though these are very complex enzymes, we have produced active hydrogenases with our cell-free methods. We are now perfecting the screening methods for rapid and accurate identification of improved enzymes. After these new enzymes are identified, the project will progress toward metabolic engineering and bioreactor design research to achieve the scales and economies required.

To address environmental needs, we are developing an improved water filters using an amazing membrane protein, Aquaporin Z. It has the ability to reject all other chemicals and ions except water. We have efficiently expressed the protein into lipid bilayer vesicles and are now working to cast these membranes on porous supports to complete the development of a new and powerful water purification technology. The same lessons will be applied toward the development of a new class of biosensors that brings high sensitivity and selectivity.

Academic Appointments


Honors & Awards


  • James H. Clark Professor in the School of Engineering, Stanford University (2008)
  • The James Bailey Award, Soc for Biol. Eng. (Am Inst. For Chem. Eng.) (2007)
  • Opening Keynote Speaker, Recent Advances in Ferm Tech (RAFT VII) (2007)
  • Keynote Lecture, 20th Meeting for the European Society for Animal Cell Tech, Dresden, Germany (2006)
  • Member, National Academy of Engineering (NAE) (2006)
  • Leland T. Edwards Professor in the School of Engineering, Stanford University (2006)
  • Elmer Gaden Award, Stanford University (2005)
  • Distinguished Alumnus Award, S.Dak. School of Mines and Technology (2005)
  • Amgen Award, Society of Industrial Microbiology (2002)
  • Opening Keynote Lecture, 2nd International Conference on Recombinant Protein Production (1999)
  • Member, National Academy of Engineering (1997)
  • Co-Chair, Biochemical Engineering X (1994)
  • Keynote Address, Biotech (2000)
  • Keynote Address, Asia-Pacific Biochemical Engineering Conference (1994)
  • James Van Lanen Service Award, Division of Biochemical Technology, American Chem. Soc. (1993)
  • Inducted as founding Fellow, The American Institute of Medical and Biological Engineers (1993)
  • Keynote Address, Inaugural Meeting of The American Institute of Medical and Biological Engineers (1992)
  • Member, National Research Council Committee on Bioprocess Engineering (1991)
  • Florasynth Fellowship, Institute of Food Technologist (1977)
  • Fellowship in Biochemical Engineering, Pfizer (1976)

Boards, Advisory Committees, Professional Organizations


  • Member, National Academy of Engineering (2013 - Present)

Professional Education


  • PhD, Massachusetts Institute of Technolog (1978)
  • ScD, MIT, Biochemical Engineering (1978)

2013-14 Courses


Postdoctoral Advisees


Journal Articles


  • Escherichia coli-based cell free production of flagellin and ordered flagellin display on virus-like particles. Biotechnology and bioengineering Lu, Y., Welsh, J. P., Chan, W., Swartz, J. R. 2013; 110 (8): 2073-2085

    Abstract

    Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this article, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300?µg/mL and the product had much higher affinity for the TLR5 receptor (EC50?=?2.4?±?1.4?pM) than previously reported. The flagellin coding sequence was first optimized for cell-free expression. We then found that the D0 domain at the C-terminus of flagellin was susceptible to proteolytic degradation in the CFPS system. Proteolysis was reduced by protease inhibitors, the use of protease-deficient cell extracts or deletion of the flagellin D0 domain. A human Toll-Like Receptor 5 (hTLR5)-specific bioactivity analysis of purified flagellin demonstrated that, although the D0 domain is far from the TLR5 recognition region, it is important for flagellin bioactivity. We next incorporated a non-natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus-like particles (VLPs) using bioorthogonal azide-alkyne cycloaddition reactions. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10-fold. Biotechnol. Bioeng. 2013;9999: XX-XX. © 2013 Wiley Periodicals, Inc.

    View details for DOI 10.1002/bit.24903

    View details for PubMedID 23519642

  • Pluripotency transcription factor Sox2 is strongly adsorbed by heparin but requires a protein transduction domain for cell internalization BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Albayrak, C., Yang, W. C., Swartz, J. R. 2013; 431 (3): 641-645

    Abstract

    The binding of protein transduction domain (PTD)-conjugated proteins to heparan sulfate is an important step in cellular internalization of macromolecules. Here, we studied the pluripotency transcription factor Sox2, with or without the nonaarginine (R9) PTD. Unexpectedly, we observed that Sox2 is strongly adsorbed by heparin and by the fibroblasts without the R9 PTD. However, only the R9Sox2 fusion protein is internalized by the cells. These results collectively show that binding to heparan sulfate is not sufficient for cellular uptake, thereby supporting a recent hypothesis that other proteins play a role in cell internalization of PTD-conjugated proteins.

    View details for DOI 10.1016/j.bbrc.2012.11.016

    View details for Web of Science ID 000315842800047

    View details for PubMedID 23318174

  • Using E. coli-based cell-free protein synthesis to evaluate the kinetic performance of an orthogonal tRNA and aminoacyl-tRNA synthetase pair BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Albayrak, C., Swartz, J. R. 2013; 431 (2): 291-295

    Abstract

    Even though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in Escherichia coli, their kinetic parameters have not been evaluated. Here we use a cell-free protein synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting synthesis of modified proteins (i.e. proteins with nnAAs). Titration experiments and estimates of turnover numbers suggest that the orthogonal synthetase is a very slow catalyst when compared to the native E. coli synthetases. The estimated k(cat) for the orthogonal synthetase specific to the nnAA p-propargyloxyphenylalanine (pPaF) is 5.4 × 10(-5) s(-1). Thus, this catalyst may be the limiting factor for nnAA incorporation when using this approach. These titration experiments also resulted in the highest reported cell-free accumulation of two different modified proteins (450 ± 20 ?g/ml CAT109pAzF and 428±2?g/ml sfGFP23pPaF) using the standard KC6 cell extract and either the PANOx SP or the inexpensive Glu NMP cell-free recipe.

    View details for DOI 10.1016/j.bbrc.2012.12.108

    View details for Web of Science ID 000315325300031

    View details for PubMedID 23291171

  • Nuclear Resonance Vibrational Spectroscopy and Electron Paramagnetic Resonance Spectroscopy of Fe-57-Enriched [FeFe] Hydrogenase Indicate Stepwise Assembly of the H-Cluster BIOCHEMISTRY Kuchenreuther, J. M., Guo, Y., Wang, H., Myers, W. K., George, S. J., Boyke, C. A., Yoda, Y., Alp, E. E., Zhao, J., Britt, R. D., Swartz, J. R., Cramer, S. P. 2013; 52 (5): 818-826

    Abstract

    The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe-S clusters that facilitate the transfer of an electron to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H(2). Here, we have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe-4S] ferredoxin as well as a model complex resembling the [2Fe](H) catalytic domain of the H-cluster. To enrich the hydrogenase with (57)Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with (56)Fe-labeled Fe-S clusters was activated in vitro using (57)Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe](H) subcluster with (57)Fe, which NRVS confirms by detecting (57)Fe-CO and (57)Fe-CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second (57)Fe-S cluster. Electron paramagnetic resonance (EPR) spectroscopy indicates that this (57)Fe-enriched metal center is not the [4Fe-4S](H) subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an (56)Fe-enriched [4Fe-4S](H) cluster during in vitro maturation, providing unambiguous evidence of stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.

    View details for DOI 10.1021/bi301336r

    View details for Web of Science ID 000314675800006

    View details for PubMedID 23249091

  • Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation. Nucleic acids research Albayrak, C., Swartz, J. R. 2013; 41 (11): 5949-63

    Abstract

    We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9-1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50-88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA(opt)) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.

    View details for PubMedID 23589624

  • Cell-free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens BIOTECHNOLOGY AND BIOENGINEERING Welsh, J. P., Lu, Y., He, X., Greenberg, H. B., Swartz, J. R. 2012; 109 (12): 2962-2969

    Abstract

    In order to effectively combat pandemic influenza threats, there is a need for more rapid and robust vaccine production methods. In this article, we demonstrate E. coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce domains from the protein hemagglutinin (HA), which is present on the surface of the influenza virus. The portion of the HA coding sequence for the "head" domain from the 2009 pandemic H1N1 strain was first optimized for E. coli expression. The protein domain was then produced in CFPS reactions and purified in soluble form first as a monomer and then as a trimer by a C-terminal addition of the T4 bacteriophage foldon domain. Production of soluble trimeric HA head domain was enhanced by introducing stabilizing amino acid mutations to the construct in order to avoid aggregation. Trimerization was verified using size exclusion HPLC, and the stabilized HA head domain trimer was more effectively recognized by antibodies from pandemic H1N1 influenza vaccine recipients than was the monomer and also bound to sialic acids more strongly, indicating that the trimers are correctly formed and could be potentially effective as vaccines.

    View details for DOI 10.1002/bit.24581

    View details for Web of Science ID 000310465300008

    View details for PubMedID 22729608

  • New Insights into [FeFe] Hydrogenase Activation and Maturase Function PLOS ONE Kuchenreuther, J. M., Britt, R. D., Swartz, J. R. 2012; 7 (9)

    Abstract

    [FeFe] hydrogenases catalyze H(2) production using the H-cluster, an iron-sulfur cofactor that contains carbon monoxide (CO), cyanide (CN(-)), and a dithiolate bridging ligand. The HydE, HydF, and HydG maturases assist in assembling the H-cluster and maturing hydrogenases into their catalytically active form. Characterization of these maturases and in vitro hydrogenase activation methods have helped elucidate steps in the H-cluster biosynthetic pathway such as the HydG-catalyzed generation of the CO and CN(-) ligands from free tyrosine. We have refined our cell-free approach for H-cluster synthesis and hydrogenase maturation by using separately expressed and purified HydE, HydF, and HydG. In this report, we illustrate how substrates and protein constituents influence hydrogenase activation, and for the first time, we show that each maturase can function catalytically during the maturation process. With precise control over the biomolecular components, we also provide evidence for H-cluster synthesis in the absence of either HydE or HydF, and we further show that hydrogenase activation can occur without exogenous tyrosine. Given these findings, we suggest a new reaction sequence for the [FeFe] hydrogenase maturation pathway. In our model, HydG independently synthesizes an iron-based compound with CO and CN(-) ligands that is a precursor to the H-cluster [2Fe](H) subunit, and which we have termed HydG-co. We further propose that HydF is a transferase that stabilizes HydG-co and also shuttles the complete [2Fe](H) subcluster to the hydrogenase, a translocation process that may be catalyzed by HydE. In summary, this report describes the first example of reconstructing the [FeFe] hydrogenase maturation pathway using purified maturases and subsequently utilizing this in vitro system to better understand the roles of HydE, HydF, and HydG.

    View details for DOI 10.1371/journal.pone.0045850

    View details for Web of Science ID 000309556100091

    View details for PubMedID 23049878

  • A vaccine directed to B cells and produced by cell-free protein synthesis generates potent antilymphoma immunity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ng, P. P., Jia, M., Patel, K. G., Brody, J. D., Swartz, J. R., Levy, S., Levy, R. 2012; 109 (36): 14526-14531

    Abstract

    Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell-targeting moiety (anti-CD19) and a lymphoma Id. This molecule (?CD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the ?CD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4(+) helper T cells. Consequently, the ?CD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.

    View details for DOI 10.1073/pnas.1211018109

    View details for Web of Science ID 000308912600051

    View details for PubMedID 22875703

  • Simplifying and streamlining Escherichia coli-based cell-free protein synthesis BIOTECHNOLOGY PROGRESS Yang, W. C., Patel, K. G., Wong, H. E., Swartz, J. R. 2012; 28 (2): 413-420

    Abstract

    Escherichia coli cell-free protein synthesis (CFPS) uses E. coli extracts to make active proteins in vitro. The basic CFPS reaction mixture is comprised of four main reagent components: (1) energy source and CFPS chemicals, (2) DNA encoding the protein of interest, (3) T7 RNA Polymerase (RNAP) for transcription, and (4) cell extract for translation. In this work, we have simplified and shortened the protocols for preparing the CFPS chemical mixture, cell extract, and T7 RNAP. First, we streamlined the workflow for preparing the CFPS chemical solutions by combining all the chemicals into a single reagent mixture, which we call Premix. We showed that productive cell extracts could be made from cells grown in simple shake flasks, and we also truncated the preparation protocol. Finally, we discovered that T7 RNAP purification was not necessary for CFPS. Crude lysate from cells over-expressing T7 RNAP could be used without deleteriously affecting protein production. Using chloramphenicol acetyltransferase (CAT) as a model protein, we showed that these streamlined protocols still support high-yielding CFPS. These simplified procedures save time and offer greater accessibility to our laboratory's CFPS technology.

    View details for DOI 10.1002/btpr.1509

    View details for Web of Science ID 000302607100013

    View details for PubMedID 22275217

  • Generation of hydrogen from NADPH using an [FeFe] hydrogenase INTERNATIONAL JOURNAL OF HYDROGEN ENERGY Smith, P. R., Bingham, A. S., Swartz, J. R. 2012; 37 (3): 2977-2983
  • Evolution of an [FeFe] hydrogenase with decreased oxygen sensitivity INTERNATIONAL JOURNAL OF HYDROGEN ENERGY Bingham, A. S., Smith, P. R., Swartz, J. R. 2012; 37 (3): 2965-2976
  • Transforming Biochemical Engineering with Cell-Free Biology AICHE JOURNAL Swartz, J. R. 2012; 58 (1): 5-13

    View details for DOI 10.1002/aic.13701

    View details for Web of Science ID 000301224400003

  • Solubility partner IF2 Domain I enables high yield synthesis of transducible transcription factors in Escherichia coli PROTEIN EXPRESSION AND PURIFICATION Yang, W. C., Welsh, J. P., Lee, J., Cooke, J. P., Swartz, J. R. 2011; 80 (1): 145-151

    Abstract

    Since the discovery that somatic cells could be reprogrammed back to a pluripotent state through the viral expression of a certain set of transcription factors, there has been great interest in reprogramming using a safer and more clinically relevant protein-based approach. However, the search for an efficient reprogramming approach utilizing the transcription factors in protein form requires a significant amount of protein material. Milligram quantities of transcription factors are challenging to obtain due to low yields and poor solubility. In this work, we describe enhanced production of the pluripotency transcription factors Oct4, Sox2, Klf4, Nanog, and Lin28 after fusing them to a solubility partner, IF2 Domain I (IF2D1). We expressed and purified milligram quantities of the fusion proteins. Though the transcription factor passenger proteins became insoluble after removal of the IF2D1, the un-cleaved Oct4, Sox2, Klf4, and Nanog fusion proteins exhibited specific binding to their consensus DNA sequences. However, when we administered the un-cleaved IF2D1-Oct4-R9 and IF2D1-Sox2-R9 to fibroblasts and measured their ability to influence transcriptional activity, we found that they were not fully bioactive; IF2D1-Oct4-R9 and IF2D1-Sox2-R9 influenced only a subset of their downstream gene targets. Thus, while the IF2D1 solubility partner enabled soluble production of the fusion protein at high levels, it did not yield fully bioactive transcription factors.

    View details for DOI 10.1016/j.pep.2011.06.017

    View details for Web of Science ID 000295608800022

    View details for PubMedID 21757009

  • Discovery of Improved EGF Agonists Using a Novel In Vitro Screening Platform JOURNAL OF MOLECULAR BIOLOGY Lui, B. H., Cochran, J. R., Swartz, J. R. 2011; 413 (2): 406-415

    Abstract

    Directed evolution is a powerful strategy for protein engineering; however, evolution of pharmaceutical proteins has been limited by the reliance of current screens on binding interactions. Here, we present a method that identifies protein mutants with improved overall cellular efficacy, an objective not feasible with previous approaches. Mutated protein libraries were produced in soluble, active form by means of cell-free protein synthesis. The efficacy of each individual protein was determined at a uniform dosage with a high-throughput protein product assay followed by a cell-based functional assay without requiring protein purification. We validated our platform by first screening mock libraries of epidermal growth factor (EGF) for stimulation of cell proliferation. We then demonstrated its effectiveness by identifying EGF mutants with significantly enhanced mitogenic activity at low concentrations compared to that of wild-type EGF. This is the first report of EGF mutants with improved biological efficacy despite much previous effort. Our platform can be extended to engineer a broad range of proteins, offering a general method to evolve proteins for improved biological efficacy.

    View details for DOI 10.1016/j.jmb.2011.08.028

    View details for Web of Science ID 000296404100010

    View details for PubMedID 21888916

  • A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA ANALYTICAL BIOCHEMISTRY Yang, W. C., Swartz, J. R. 2011; 415 (2): 168-174

    Abstract

    We present a rapid method for quantifying the apparent DNA binding affinity and capacity of recombinant transcription factors (TFs). We capture His6-tagged TFs using nickel-nitrilotriacetic acid (Ni-NTA) agarose and incubate the immobilized TFs with fluorescently labeled cognate DNA probes. After washing, the strength of the fluorescence signal indicates the extent of DNA binding. The assay was validated using two pluripotency-regulating TFs: SOX2 and NANOG. Using competitive binding analysis with nonlabeled competitor DNA, we show that SOX2 and NANOG specifically bind to their consensus sequences. We also determined the apparent affinity of SOX2 and NANOG for their consensus sequences to be 54.2±9 and 44.0±6nM, respectively, in approximate agreement with literature values. Our assay does not require radioactivity, but radioactively labeling the TFs enables the measurement of absolute amounts of immobilized SOX2 and NANOG and, hence, a DNA-to-protein binding ratio. SOX2 possesses a 0.95 DNA-to-protein binding ratio, whereas NANOG possesses a 0.44 ratio, suggesting that most of the SOX2 and approximately half of the NANOG are competent for DNA binding. Alternatively, the NANOG dimer may be capable of binding only one DNA target. This flexible DNA binding assay enables the analysis of crude or purified samples with or without radioactivity.

    View details for DOI 10.1016/j.ab.2011.03.027

    View details for Web of Science ID 000291904700010

    View details for PubMedID 21447317

  • Localization of BiP to Translating Ribosomes Increases Soluble Accumulation of Secreted Eukaryotic Proteins in an Escherichia Coli Cell-Free System BIOTECHNOLOGY AND BIOENGINEERING Welsh, J. P., Bonomo, J., Swartz, J. R. 2011; 108 (8): 1739-1748

    Abstract

    The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS.

    View details for DOI 10.1002/bit.23111

    View details for Web of Science ID 000292602600001

    View details for PubMedID 21351069

  • Efficient disulfide bond formation in virus-like particles JOURNAL OF BIOTECHNOLOGY Bundy, B. C., Swartz, J. R. 2011; 154 (4): 230-239

    Abstract

    Virus-like particles (VLPs) consist of a virus's outer shell but without the genome. Similar to the virus, VLPs are monodisperse nano-capsules which have a known morphology, maintain a high degree of symmetry, and can be engineered to encapsidate the desired cargo. VLPs are of great interest for vaccination, drug/gene delivery, imaging, sensing, and material science applications. Here we demonstrate the ability to control the disulfide bond formation in VLPs by directly controlling the redox potential during or after production and assembly of VLPs. The open cell-free protein synthesis environment, which has been reported to produce VLPs at yields comparable or greater than traditional in vivo technologies, was employed. Optimal conditions for disulfide bond formation were found to be VLP dependent, and a cooperative effect in the formation of such bonds was observed.

    View details for DOI 10.1016/j.jbiotec.2011.04.011

    View details for Web of Science ID 000292958600007

    View details for PubMedID 21536082

  • Cell-free H-cluster Synthesis and [FeFe] Hydrogenase Activation: All Five CO and CN- Ligands Derive from Tyrosine PLOS ONE Kuchenreuther, J. M., George, S. J., Grady-Smith, C. S., Cramer, S. P., Swartz, J. R. 2011; 6 (5)

    Abstract

    [FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN?) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN? ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

    View details for DOI 10.1371/journal.pone.0020346

    View details for Web of Science ID 000291097600048

    View details for PubMedID 21673792

  • Surface Functionalization of Virus-Like Particles by Direct Conjugation Using Azide-Alkyne Click Chemistry BIOCONJUGATE CHEMISTRY Patel, K. G., Swartz, J. R. 2011; 22 (3): 376-387

    Abstract

    We present a cell-free protein synthesis (CFPS) platform and a one-step, direct conjugation scheme for producing virus-like particle (VLP) assemblies that display multiple ligands including proteins, nucleic acids, and other molecules. Using a global methionine replacement approach, we produced bacteriophage MS2 and bacteriophage Q? VLPs with surface-exposed methionine analogues (azidohomoalanine and homopropargylglycine) containing azide and alkyne side chains. CFPS enabled the production of VLPs with yields of ~ 300 ?g/mL and with 85% incorporation of methionine analogues without requiring a methionine auxotrophic production host. We then directly conjugated azide- and alkyne-containing proteins (including an antibody fragment and the granulocyte-macrophage colony stimulating factor, or GM-CSF), nucleic acids and poly(ethylene glycol) chains to the VLP surface using Cu(I) catalyzed click chemistry. The GM-CSF protein, after conjugation to VLPs, was shown to partially retain its ability to stimulate the proliferation of cells. Conjugation of GM-CSF to VLPs resulted in a 3-5-fold reduction in its bioactivity. The direct attachment scheme facilitated conjugation of three different ligands to the VLPs in a single step, and enabled control of the relative ratios and surface abundance of the attached species. This platform can be used for the production of novel VLP bioconjugates for use as drug delivery vehicles, diagnostics, and vaccines.

    View details for DOI 10.1021/bc100367u

    View details for Web of Science ID 000288401400009

    View details for PubMedID 21355575

  • Escherichia coli-based production of a tumor idiotype antibody fragment - tetanus toxin fragment C fusion protein vaccine for B cell lymphoma PROTEIN EXPRESSION AND PURIFICATION Patel, K. G., Ng, P. P., Levy, S., Levy, R., Swartz, J. R. 2011; 75 (1): 15-20

    Abstract

    The unique immunoglobulin idiotype expressed on the surface of B lymphoma cells can be used as an effective antigen in tumor-specific vaccines when fused to immunostimulatory proteins and cytokines. A DNA vaccine encoding for an idiotype antibody single chain Fv (scFv) fragment fused to the Tetanus Toxin Fragment C (TTFrC) has been shown to induce protective anti-tumor responses. Protein-based strategies may be more desirable since they provide greater control over dosage, duration of exposure, and in vivo distribution of the vaccine. However, production of fusion protein vaccines containing complex disulfide bonded idiotype antibodies and antibody-derived fragments is challenging. We use an Escherichia coli-based cell-free protein synthesis platform as well as high-level expression of E. coli inclusion bodies followed by refolding for the rapid generation of an antibody fragment - TTFrC fusion protein vaccine. Vaccine proteins produced using both methods were shown to elicit anti-tumor humoral responses as well as protect from tumor challenge in an established B cell lymphoma mouse model. The development of technologies for the rapid production of effective patient-specific tumor idiotype-based fusion protein vaccines provides opportunities for clinical application.

    View details for DOI 10.1016/j.pep.2010.09.005

    View details for Web of Science ID 000284452600002

    View details for PubMedID 20851769

  • Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases PLOS ONE Stapleton, J. A., Swartz, J. R. 2010; 5 (12)

    Abstract

    [FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible.Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library.[FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy.

    View details for DOI 10.1371/journal.pone.0015275

    View details for Web of Science ID 000284995300026

    View details for PubMedID 21151915

  • High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli PLOS ONE Kuchenreuther, J. M., Grady-Smith, C. S., Bingham, A. S., George, S. J., Cramer, S. P., Swartz, J. R. 2010; 5 (11)

    Abstract

    The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases.In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms.The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

    View details for DOI 10.1371/journal.pone.0015491

    View details for Web of Science ID 000284572000030

    View details for PubMedID 21124800

  • Comparing the functional properties of the Hsp70 chaperones, DnaK and BiP BIOPHYSICAL CHEMISTRY Bonomo, J., Welsh, J. P., Manthiram, K., Swartz, J. R. 2010; 149 (1-2): 58-66

    Abstract

    The Hsp70 family of molecular chaperones is an essential class of chaperones that is present in many different cell types and cellular compartments. We have compared the bioactivities of the prokaryotic cytosolic Hsp70, DnaK, to that of the eukaryotic Hsp70, BiP, located in the endoplasmic reticulum (ER). Both chaperones helped to prevent protein aggregation. However, only DnaK provided enhanced refolding of denatured proteins. We also tested chaperone folding assistance during translation in the context of cell-free protein synthesis reactions for several protein targets and show that both DnaK and BiP can provide folding assistance under these conditions. Our results support previous reports suggesting that DnaK provides both post-translational and co-translational folding assistance while BiP predominantly provides folding assistance that is contemporaneous with translation.

    View details for DOI 10.1016/j.bpc.2010.04.001

    View details for Web of Science ID 000278427300008

    View details for PubMedID 20435400

  • A Cell-Free Microtiter Plate Screen for Improved [FeFe] Hydrogenases PLOS ONE Stapleton, J. A., Swartz, J. R. 2010; 5 (5)

    Abstract

    [FeFe] hydrogenase enzymes catalyze the production and dissociation of H(2), a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including their extreme oxygen sensitivity. Directed evolution has been used to improve the characteristics of a range of natural catalysts, but has been largely unsuccessful for [FeFe] hydrogenases because of a lack of convenient screening platforms.Here we describe an in vitro screening technology for oxygen-tolerant and highly active [FeFe] hydrogenases. Despite the complexity of the protocol, we demonstrate a level of reproducibility that allows moderately improved mutants to be isolated. We have used the platform to identify a mutant of the Chlamydomonas reinhardtii [FeFe] hydrogenase HydA1 with a specific activity approximately 4 times that of the wild-type enzyme.Our results demonstrate the feasibility of using the screen presented here for large-scale efforts to identify improved biocatalysts for energy applications. The system is based on our ability to activate these complex enzymes in E. coli cell extracts, which allows unhindered access to the protein maturation and assay environment.

    View details for DOI 10.1371/journal.pone.0010554

    View details for Web of Science ID 000277467900010

    View details for PubMedID 20479937

  • Site-Specific Incorporation of p-Propargyloxyphenylalanine in a Cell-Free Environment for Direct Protein-Protein Click Conjugation BIOCONJUGATE CHEMISTRY Bundy, B. C., Swartz, J. R. 2010; 21 (2): 255-263

    Abstract

    The tyrosine analog p-propargyloxyphenylalanine (pPa), like tyrosine, has limited water solubility. It has been postulated that this limited solubility has contributed to reduced cellular uptake of pPa and thus reduced in vivo incorporation of pPa into proteins. Using a cell-free protein synthesis system (CFPS) to circumvent cellular uptake, pPa has been incorporated site-specifically into proteins with high specificity at yields up to 27 times greater than the highest previously reported yield. The alkyne group present on proteins incorporated with pPa provides a reactive residue for site-specific bioconjugation with the copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition (CuAAC). Previously, incorporation of another CuAAC-compatible unnatural amino acid p-azido-l-phenylalanine (pAz) was demonstrated with CFPS. However, incorporation of pPa may be preferred over pAz due to the instability of the pAz's aryl-azido moiety upon UV or near-UV light exposure. Also, the ability to incorporate site-specifically both reactants of the CuAAC (the alkyne group of pPa and the azido group of pAz) into proteins enables direct site-specific conjugation of heterologous proteins. We have demonstrated (for the first time to our knowledge) a one-step, linker-less, site-specific, direct protein-to-protein conjugation using CuAAC and unnatural amino acids.

    View details for DOI 10.1021/bc9002844

    View details for Web of Science ID 000274514300009

    View details for PubMedID 20099875

  • Cell-free production of Gaussia princeps luciferase - antibody fragment bioconjugates for ex vivo detection of tumor cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Patel, K. G., Ng, P. P., Kuo, C., Levy, S., Levy, R., Swartz, J. R. 2009; 390 (3): 971-976

    Abstract

    Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

    View details for DOI 10.1016/j.bbrc.2009.10.087

    View details for Web of Science ID 000272516700113

    View details for PubMedID 19852937

  • Cell-Free Production of Transducible Transcription Factors for Nuclear Reprogramming BIOTECHNOLOGY AND BIOENGINEERING Yang, W. C., Patel, K. G., Lee, J., Ghebremariam, Y. T., Wong, H. E., Cooke, J. P., Swartz, J. R. 2009; 104 (6): 1047-1058

    Abstract

    Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target.

    View details for DOI 10.1002/bit.22517

    View details for Web of Science ID 000273813400001

    View details for PubMedID 19718703

  • Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Welsh, J. P., Patel, K. G., Manthiram, K., Swartz, J. R. 2009; 389 (4): 563-568

    Abstract

    Gaussia luciferase (GLuc) from the copepod Gaussia princeps is both the smallest and brightest known luciferase. GLuc catalyzes the oxidation of coelenterazine to produce an intense blue light but with a very short emission half-life. We report mutated GLucs with much longer luminescence half-lives that retain the same initial intensity as the wild-type enzyme. The GLuc variants were produced using cell-free protein synthesis to provide high yields and rapid production of fully active product as well as simple non-natural amino acid substitution. By incorporating homopropargylglycine and attaching PEG using azide-alkyne click reactions, we also show that the four methionines in GLuc are surface accessible. The mutants provide a significantly improved reporter protein for both in vivo and in vitro studies, and the successful non-natural amino acid incorporation and PEG attachment indicate the feasibility of producing useful bioconjugates using click attachment reactions.

    View details for DOI 10.1016/j.bbrc.2009.09.006

    View details for Web of Science ID 000271151100001

    View details for PubMedID 19825431

  • Tyrosine, Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation PLOS ONE Kuchenreuther, J. M., Stapleton, J. A., Swartz, J. R. 2009; 4 (10)

    Abstract

    [FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H(2). These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe-6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated.We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation.The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives.

    View details for DOI 10.1371/journal.pone.0007565

    View details for Web of Science ID 000271147200008

    View details for PubMedID 19855833

  • Cell-Free Synthesis of Functional Aquaporin Z in Synthetic Liposomes BIOTECHNOLOGY AND BIOENGINEERING Hovijitra, N. T., Wuu, J. J., Peaker, B., Swartz, J. R. 2009; 104 (1): 40-49

    Abstract

    The challenges involved in producing sufficient quantities of aquaporins for precise biophysical characterization have limited our knowledge of this important class of molecules. This article describes a cell-free protein synthesis method for producing high concentrations of the E. coli water transporter, aquaporin Z (AqpZ), in synthetic liposomes. To our knowledge, this is the first report of in vitro synthesis of a membrane protein directly into synthetic liposomes with verified function, (i.e., transport activity and selectivity). Titration of DOPC lipid vesicles added to the cell-free reaction show that production yields of active AqpZ are dependent on the concentration of DOPC lipid vesicles added to the cell-free reaction, with 224 +/- 24 lipids required per aquaporin monomer. Supplementation of the signal recognition particle receptor (FtsY) to the cell-free reaction increases production of vesicle-associated AqpZ but not active AqpZ. Cell-free reactions using 7 mg/mL lipids that were not supplemented with FtsY produced 507 +/- 11 microg/mL of vesicle-associated AqpZ that exhibited a specific water transport activity of (2.2 +/- 0.3) x 10(-14) cm(3) s(-1) monomer(-1). Proteinase K protection, activation energy determination, and selectivity against glycerol and urea transport also confirmed the production of correctly folded AqpZ. This technique is capable of producing milligram quantities of aquaporin that can be readily assayed for function, facilitating biophysical characterization and high-throughput analysis.

    View details for DOI 10.1002/bit.22385

    View details for Web of Science ID 000269096800006

    View details for PubMedID 19557835

  • Universal cell-free protein synthesis NATURE BIOTECHNOLOGY Swartz, J. R. 2009; 27 (8): 731-732

    View details for DOI 10.1038/nbt0809-731

    View details for Web of Science ID 000268774500021

    View details for PubMedID 19668180

  • High-Level Cell-Free Synthesis Yields of Proteins Containing Site-Specific Non-Natural Amino Acids BIOTECHNOLOGY AND BIOENGINEERING Goerke, A. R., Swartz, J. R. 2009; 102 (2): 400-416

    Abstract

    We describe an E. coli-based cell-free system for the production of proteins with a non-natural amino acid (nnAA) incorporated site-specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNA(Tyr) pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200-300 microg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m(2)/mL. This new E. coli-based cell-free procedure produced up to 400 microg/mL of eCAT109pAz, 660 microg/mL of eDHFR10pAz, and 210 microg/mL of mDHFR31pAz with p-azido-L-phenylalanine (pAz) incorporated site-specifically at the amber nonsense codon. O-methyl-L-tyrosine and p-acetyl-L-phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell-free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)-catalyzed (3 + 2) cycloadditions (click chemistry).

    View details for DOI 10.1002/bit.22070

    View details for Web of Science ID 000262540300008

    View details for PubMedID 18781689

  • Continued Protein Synthesis at Low [ATP] and [GTP] Enables Cell Adaptation during Energy Limitation JOURNAL OF BACTERIOLOGY Jewett, M. C., Miller, M. L., Chen, Y., Swartz, J. R. 2009; 191 (3): 1083-1091

    Abstract

    One of biology's critical ironies is the need to adapt to periods of energy limitation by using the energy-intensive process of protein synthesis. Although previous work has identified the individual energy-requiring steps in protein synthesis, we still lack an understanding of the dependence of protein biosynthesis rates on [ATP] and [GTP]. Here, we used an integrated Escherichia coli cell-free platform that mimics the intracellular, energy-limited environment to show that protein synthesis rates are governed by simple Michaelis-Menten dependence on [ATP] and [GTP] (K(m)(ATP), 27 +/- 4 microM; K(m)(GTP), 14 +/- 2 microM). Although the system-level GTP affinity agrees well with the individual affinities of the GTP-dependent translation factors, the system-level K(m)(ATP) is unexpectedly low. Especially under starvation conditions, when energy sources are limited, cells need to replace catalysts that become inactive and to produce new catalysts in order to effectively adapt. Our results show how this crucial survival priority for synthesizing new proteins can be enforced after rapidly growing cells encounter energy limitation. A diminished energy supply can be rationed based on the relative ATP and GTP affinities, and, since these affinities for protein synthesis are high, the cells can adapt with substantial changes in protein composition. Furthermore, our work suggests that characterization of individual enzymes may not always predict the performance of multicomponent systems with complex interdependencies. We anticipate that cell-free studies in which complex metabolic systems are activated will be valuable tools for elucidating the behavior of such systems.

    View details for DOI 10.1128/JB.00852-08

    View details for Web of Science ID 000262609200045

    View details for PubMedID 19028899

  • An integrated cell-free metabolic platform for protein production and synthetic biology MOLECULAR SYSTEMS BIOLOGY Jewett, M. C., Calhoun, K. A., Voloshin, A., Wuu, J. J., Swartz, J. R. 2008; 4

    Abstract

    Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.

    View details for DOI 10.1038/msb.2008.57

    View details for Web of Science ID 000260722900002

    View details for PubMedID 18854819

  • Escherichia coli-based cell-free synthesis of virus-like particles BIOTECHNOLOGY AND BIOENGINEERING Bundy, B. C., Franciszkowicz, M. J., Swartz, J. R. 2008; 100 (1): 28-37

    Abstract

    Virus-like particles (VLP) have received considerable attention for vaccine, drug delivery, gene therapy and material science applications. Although the number of unique VLP and their applications are rapidly growing, the positive impact of VLP applications is limited by the current diverse, expensive, and typically low-yielding production technologies available. These technologies, when scaled, often result in structurally and compositionally inconsistent products. We present Escherichia coli-based cell-free protein synthesis as a production technology to overcome many of the limitations of current VLP production processes. Using this technique, the MS2 bacteriophage coat protein VLP was produced at a yield 14 times the best published production yield. Also, a C-terminally truncated Hepatitis B core protein VLP was produced at similarly high yields (6 x 10(13) VLP/mL). These VLP were found to have comparable characteristics to those produced in vivo. The scalability of this technology was tested without loss in production yields. To our knowledge, this is the first time a prokaryote-based in vitro transcription/translation system has generated a virus-like particle.

    View details for DOI 10.1002/bit.21716

    View details for Web of Science ID 000254786200003

    View details for PubMedID 18023052

  • High yield cell-free production of integral membrane proteins without refolding or detergents BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Wuu, J. J., Swartz, J. R. 2008; 1778 (5): 1237-1250

    Abstract

    Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

    View details for DOI 10.1016/j.bbamem.2008.01.023

    View details for Web of Science ID 000256211700005

    View details for PubMedID 18295592

  • Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase METABOLIC ENGINEERING Goerke, A. R., Loening, A. M., Gambhir, S. S., Swartz, J. R. 2008; 10 (3-4): 187-200

    Abstract

    Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412mug/mL of purified GLuc, relative to 5mug/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2x10(24)photons/s/mol, the highest reported activity for any characterized luciferase.

    View details for DOI 10.1016/j.ymben.2008.04.001

    View details for Web of Science ID 000261595600006

    View details for PubMedID 18555198

  • Development of cell-free protein synthesis platforms for disulfide bonded proteins BIOTECHNOLOGY AND BIOENGINEERING Goerke, A. R., Swartz, J. R. 2008; 99 (2): 351-367

    Abstract

    The use of cell-free protein synthesis (CFPS) for recombinant protein production is emerging as an important technology. For example, the openness of the cell-free system allows control of the reaction environment to promote folding of disulfide bonded proteins in a rapid and economically feasible format. These advantages make cell-free protein expression systems particularly well suited for producing patient specific therapeutic vaccines or antidotes in response to threats from natural and man-made biological agents and for pharmaceutical proteins that are difficult to produce in living cells. In this work we assess the versatility of modern cell-free methods, optimize expression and folding parameters, and highlight the importance of rationally designed plasmid templates for producing mammalian secreted proteins, fusion proteins, and antibody fragments in our E. coli-based CFPS system. Two unique CFPS platforms were established by developing standardized extract preparation protocols and generic cell-free reaction conditions. Generic reaction conditions enabled all proteins to express well with the best therapeutic protein yield at 710 microg/mL, an antibody fragment at 230 microg/mL, and a vaccine fusion protein at 300 microg/mL; with the majority correctly folded. Better yields were obtained when cell-free reaction conditions were optimized for each protein. Establishing general CFPS platforms enhances the potential for cell-free protein synthesis to reliably produce complex protein products at low production and capital costs with very rapid process development timelines.

    View details for DOI 10.1002/bit.21567

    View details for Web of Science ID 000252270300012

    View details for PubMedID 17626291

  • Cell-free synthesis and maturation of [FeFe] hydrogenases BIOTECHNOLOGY AND BIOENGINEERING Boyer, M. E., Stapleton, J. A., Kuchenreuther, J. M., Wang, C., Swartz, J. R. 2008; 99 (1): 59-67

    Abstract

    [FeFe] hydrogenases catalyze the reversible reduction of protons to molecular hydrogen (Adams (1990); Biochim Biophys Acta 1020(2): 115-145) and are of significant interest for the biological production of hydrogen fuel. They are complex proteins with active sites containing iron, sulfur, and carbon monoxide and cyanide ligands (Peters et al. (1998); Science 282(5395): 1853-1858). Maturation enzymes for [FeFe] hydrogenases have been identified (Posewitz et al. (2004); J Biol Chem 279(24): 25711-25720), but complete mechanisms have not yet been elucidated. The study of [FeFe] hydrogenases has been impeded by the lack of an easily manipulated expression/activation system capable of producing these complex and extremely oxygen-sensitive enzymes. Here we show the first expression of functional [FeFe] hydrogenases in an Escherichia coli-based cell-free transcription/translation system. We have produced and matured both algal and bacterial hydrogenases using E. coli cell extracts containing the HydG, HydE, and HydF proteins from Shewanella oneidensis. The current system produces approximately 22 microg/mL of active protein, constituting approximately 44% of the total protein produced. Active protein yield is greatly enhanced by pre-incubation of the maturation enzyme-containing extract with inorganic iron and sulfur for reconstitution of the [Fe-S] clusters in HydG, HydE, and HydF. The absence of cell walls permits direct addition of cofactors and substrates, enabling rapid production of active protein and providing control over the maturation conditions. These new capabilities will enhance the investigation of complex proteins requiring helper proteins for maturation and move us closer to the development of improved hydrogenases for biological production of hydrogen as a clean, renewable alternative fuel.

    View details for DOI 10.1002/bit.21511

    View details for Web of Science ID 000251639800007

    View details for PubMedID 17546685

  • A sequential expression system for high-throughput functional genomic analysis PROTEOMICS Woodrow, K. A., Swartz, J. R. 2007; 7 (21): 3870-3879

    Abstract

    A method employing sequential rounds of cell-free protein synthesis (CFPS) was developed to identify gene products influencing the complex metabolic systems that result in protein accumulation and folding in vitro. The first round of CFPS creates an array of cell extracts individually enriched with a single gene product expressed in-parallel from linear DNA expression templates (ETs). The cell extract is engineered to enhance template stability and to provide reaction conditions conducive for general protein activation. Following first-round expression, linear templates are selectively degraded and a plasmid template for a reporter enzyme is added to initiate a subsequent round of protein expression. Reporter concentration and activity identify first-round gene products that affect amino acid and nucleic acid stability, energy supply, protein expression, stability, and activation. This sequential CFPS system provides a unique format for the functional genomic identification of broadly diverse metabolic activities.

    View details for DOI 10.1002/pmic.200700471

    View details for Web of Science ID 000251016800005

    View details for PubMedID 17960738

  • Cell-free synthesis of proteins that require disulfide bonds using glucose as an energy source BIOTECHNOLOGY AND BIOENGINEERING Knapp, K. G., Goerke, A. R., Swartz, J. R. 2007; 97 (4): 901-908

    Abstract

    The primary objective of this work was to create a cell-free protein synthesis extract that produces proteins requiring disulfide bonds while using glucose as an energy source. We attempted to avoid using iodoacetamide (IAM) to stabilize the required oxidizing thiol redox potential, since previous IAM pretreatments prevented glucose utilization apparently by inactivating glyceraldehyde 3-phosphate dehydrogenase (G-3PDH). Instead, the glutathione reductase (Gor)-mediated disulfide reductase system was disabled by deleting the gor gene from the KC6 cell-extract source strain. The thioredoxin reductase (TrxB)-mediated system was disabled by first adding a purification tag to the trxB gene in the chromosome to create strain KGK10 and then by affinity removal of the tagged TrxB. This was expected to result in a cell extract devoid of all disulfide reductase activity, but this was not the case. Although the concentration of IAM required to stabilize oxidized glutathione in the KGK10 extract could be reduced 20-fold, IAM pretreatment was still required to avoid disulfide reduction. Nonetheless, active urokinase and murine granulocyte macrophage-colony stimulating factor (mGM-CSF) were produced in reactions with KGK10 extract either with affinity removal of TrxB or with 50 microM IAM pretreatment. With the less intensive IAM pretreatment, glucose could be used as an energy source in a production system that promotes oxidative protein folding. This new protocol offers an economically feasible cell-free system for the production of secreted mammalian proteins as human therapeutics or vaccines.

    View details for DOI 10.1002/bit.21296

    View details for Web of Science ID 000247158800024

    View details for PubMedID 17154312

  • Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines BLOOD Kanter, G., Yang, J., Voloshin, A., Levy, S., Swartz, J. R., Levy, R. 2007; 109 (8): 3393-3399

    Abstract

    The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

    View details for DOI 10.1182/blood-2006-07-030593

    View details for Web of Science ID 000245658500047

    View details for PubMedID 17164345

  • Evidence for an additional disulfide reduction pathway in Escherichia coli JOURNAL OF BIOSCIENCE AND BIOENGINEERING Knapp, K. G., Swartz, J. R. 2007; 103 (4): 373-376

    Abstract

    An Escherichia coli cell-free protein synthesis cell extract has been created that lacks all known cytoplasmic disulfide reduction pathways but still retains significant reductase activity. Oxidized glutathione was partially stabilized by deleting the gene for glutathione reductase. To avoid previously reported AhpC mutations, thioredoxin reductase was only removed after extract preparation. The trxB gene was extended to encode a hemagglutinin tag so that TrxB could be removed by affinity adsorption. However, significant glutathione reductase activity remained. The unknown glutathione reductase pathway is disabled by iodoacetamide, is inhibited by NADH, and appears to use NADPH as an electron source.

    View details for DOI 10.1263/jbb.103.373

    View details for Web of Science ID 000247123800014

    View details for PubMedID 17502280

  • Energy systems for ATP regeneration in cell-free protein synthesis reactions. Methods in molecular biology (Clifton, N.J.) Calhoun, K. A., Swartz, J. R. 2007; 375: 3-17

    Abstract

    Supplying energy for cell-free protein synthesis reactions is one of the biggest challenges to the success of these systems. Oftentimes, short reaction duration is attributed to an unstable energy source. Traditional cell-free reactions use a compound with a high-energy phosphate bond, such as phosphoenolpyruvate, to generate the ATP required to drive transcription and translation. However, recent work has led to better understanding and activation of the complex metabolism that can occur during cell-free reactions. We are now able to generate ATP using energy sources that are less expensive and more stable. These energy sources generally involve multistep enzymatic reactions or recreate entire energy-generating pathways, such as glycolysis and oxidative phosphorylation. We describe the various types of energy sources used in cell-free reactions, give examples of the major classes, and demonstrate protocols for successful use of three recently developed energy systems: PANOxSP, cytomim, and glucose.

    View details for PubMedID 17634594

  • Rapid expression of functional genomic libraries JOURNAL OF PROTEOME RESEARCH Woodrow, K. A., Airen, I. O., Swartz, J. R. 2006; 5 (12): 3288-3300

    Abstract

    Genomic-scale analysis of protein function is currently limited by the ability to rapidly express the enormous diversity of protein targets in their active form. We describe a method to construct transcriptionally active expression templates (ETs) in parallel using a single PCR step wherein the overlap-extension reaction for addition of transcription regulatory elements is separated from the amplification of the full-length product by using a GC-rich single primer. Over 90% of 55 diverse genomic targets were extended with T7 regulatory elements to form ETs in high yield and purity. The unpurified ETs directed protein expression using a cell-free protein synthesis (CFPS) system supplemented with cofactors and metal ions to activate a variety of enzymes. Higher activities were obtained in the modified CFPS reactions compared to standard reaction conditions. Protein purification was avoided because the expressed enzyme activity was significantly greater than the background activity associated with the cell extract. These improvements in the parallel synthesis of linear ETs combined with enhanced in vitro enzyme activation help to make CFPS systems more attractive platforms for high-throughput evaluation of protein function.

    View details for DOI 10.1021/pr050459y

    View details for Web of Science ID 000242427800007

    View details for PubMedID 17137330

  • Effects of growth rate on cell extract performance in cell-free protein synthesis BIOTECHNOLOGY AND BIOENGINEERING Zawada, J., Swartz, J. 2006; 94 (4): 618-624

    Abstract

    Cell-free protein synthesis is a useful research tool and now stands poised to compete with in vivo expression for commercial production of proteins. However, both the extract preparation and protein synthesis procedures must be scaled up. A key challenge is producing the required amount of biomass that also results in highly active cell-free extracts. In this work, we show that the growth rate of the culture dramatically affects extract performance. Extracts prepared from cultures with a specific growth rate of 0.7/h or higher produced approximately 0.9 mg/mL of chloramphenicol acetyl transferase (CAT) in a batch reaction. In contrast, when the source culture growth rate was 0.3/h, the resulting extract produced only 0.5 mg/mL CAT. Examination of the ribosome content in the extracts revealed that the growth rate of the source cells strongly influenced the final ribosome concentration. Polysome analysis of cell-free protein synthesis reactions indicated that about 22% of the total 70S ribosomes are in polysomes for all extracts regardless of growth rate. Furthermore, the overall specific production from the 70S ribosomes is about 22 CAT proteins per ribosome over the course of the reaction in all cases. It appears that rapid culture growth rates are essential for producing a productive extract. However, growth rate does not seem to influence specific ribosome activity. Rather, the increase in extract productivity is a result of a higher ribosome concentration. These results are important for cell-free technology and also suggest an assay for intrinsic in vivo protein synthesis activity.

    View details for DOI 10.1002/bit.20831

    View details for Web of Science ID 000238350700002

    View details for PubMedID 16673418

  • Total amino acid stabilization during cell-free protein synthesis reactions JOURNAL OF BIOTECHNOLOGY Calhoun, K. A., Swartz, J. R. 2006; 123 (2): 193-203

    Abstract

    Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75-250% relative to cell-free expression using the control extract.

    View details for DOI 10.1016/j.jbiotec.2005.11.011

    View details for Web of Science ID 000237881800008

    View details for PubMedID 16442654

  • Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free system BIOTECHNOLOGY AND BIOENGINEERING Boyer, M. E., Wang, C. W., Swartz, J. R. 2006; 94 (1): 128-138

    Abstract

    The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.

    View details for DOI 10.1002/bit.20830

    View details for Web of Science ID 000237216800013

    View details for PubMedID 16570319

  • Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis BIOTECHNOLOGY AND BIOENGINEERING Underwood, K. A., Swartz, J. R., Puglisi, J. D. 2005; 91 (4): 425-435

    Abstract

    Cell-free protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. CFPS optimization efforts have focused primarily on energy supply and small molecule metabolism, though little is known about the protein synthesis machinery or what limits protein synthesis rates. Here, quantitative polysome profile analysis was used to characterize cell-free translation, thereby elucidating many kinetic parameters. The ribosome concentration in Escherichia coli-based CFPS reactions was 1.6 +/- 0.1 microM, with 72 +/- 4% actively translating at maximal protein synthesis rate. A translation elongation rate of 1.5 +/- 0.2 amino acids per second per ribosome and an initiation rate of 8.2 x 10(-9) +/- 0.3 x 10(-9) M/s, which correlates to, on average, one initiation every 60 +/- 9 s per mRNA, were determined. The measured CFPS initiation and elongation rates are an order of magnitude lower than the in vivo rates and further analysis identified elongation as the major limitation. Adding purified elongation factors (EFs) to CFPS reactions increased the ribosome elongation rate and protein synthesis rates and yields, as well as the translation initiation rate, indicating a possible coupling between initiation and elongation. Further examination of translation initiation in the cell-free system showed that the first initiation on an mRNA is slower than subsequent initiations. Our results demonstrate that polysome analysis is a valid tool to characterize cell-free translation and to identify limiting steps, that dilution of translation factors is a limitation of CFPS, and that CFPS is a useful platform for making novel observations about translation.

    View details for DOI 10.1002/bit.20529

    View details for Web of Science ID 000230915400004

    View details for PubMedID 15991235

  • Efficient and scalable method for scaling up cell free protein synthesis in batch mode BIOTECHNOLOGY AND BIOENGINEERING Voloshin, A. M., Swartz, J. R. 2005; 91 (4): 516-521

    Abstract

    A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 microL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 microL reaction volume, the PANOx SP system produced 560 +/- 36 microg/mL of active CAT and 99 +/- 10 microg/mL of active GLH protein using the new thin film approach whereas 500 microL test tube reactions produced 250 +/- 42 microg/mL and 72 +/- 7 microg/mL of active CAT and GLH respectively. Similarly, 500 microL cell-free synthesis reactions with the Cytomim system produced 481 +/- 38 microg/mL of active CAT and 109 +/- 15 microg/mL active GLH respectively in thin films compared to 29 +/- 7 microg/mL of active CAT and 5 +/- 2 microg/mL of active GLH protein in 500 microL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume.

    View details for Web of Science ID 000230915400014

    View details for PubMedID 15937883

  • An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates BIOTECHNOLOGY PROGRESS Calhoun, K. A., Swartz, J. R. 2005; 21 (4): 1146-1153

    Abstract

    Cell-free protein synthesis reactions have not been seriously considered as a viable method for commercial protein production mainly because of high reagent costs and a lack of scalable technologies. Here we address the first issue by presenting a cell-free protein synthesis system with comparable protein yields that removes the most expensive substrates and lowers the cell-free reagent cost by over 75% (excluding extract, polymerase, and plasmid) while maintaining high energy levels. This system uses glucose as the energy source and nucleoside monophosphates (NMPs) in place of nucleoside triphosphates (NTPs) as the nucleotide source. High levels of nucleoside triphosphates are generated from the monophosphates within 20 min, and the subsequent energy charge is similar in reactions beginning with either NTPs or NMPs. Furthermore, significant levels (>0.2 mM) of all NTPs are still available at the end of a 3-h incubation, and the total nucleotide pool is stable throughout the reaction. The glucose/NMP reaction was scaled up to milliliter scale using a thin film approach. Significant yields of active protein were observed for two proteins of vastly different size: chloramphenicol acetyl transferase (CAT, 25 kDa) and beta-galactosidase (472 kDa). The glucose/NMP cell-free reaction system dramatically reduces reagent costs while supplying high protein yields.

    View details for DOI 10.1021/bp050052y

    View details for Web of Science ID 000231126800018

    View details for PubMedID 16080695

  • Energizing cell-free protein synthesis with glucose metabolism BIOTECHNOLOGY AND BIOENGINEERING Calhoun, K. A., Swartz, J. R. 2005; 90 (5): 606-613

    Abstract

    In traditional cell-free protein synthesis reactions, the energy source (typically phosphoenolpyruvate (PEP) or creatine phosphate) is the most expensive substrate. However, for most biotechnology applications glucose is the preferred commercial substrate. Previous attempts to use glucose in cell-free protein synthesis reactions have been unsuccessful. We have now developed a cell-free protein synthesis reaction where PEP is replaced by either glucose or glucose-6-phosphate (G6P) as the energy source, thus allowing these reactions to compete more effectively with in vivo protein production technologies. We demonstrate high protein yields in a simple batch-format reaction through pH control and alleviation of phosphate limitation. G6P reactions can produce high protein levels ( approximately 700 microg/mL of chloramphenical acetyl transferase (CAT)) when pH is stabilized through replacement of the HEPES buffer with Bis-Tris. Protein synthesis with glucose as an energy source is also possible, and CAT yields of approximately 550 mug/mL are seen when both 10 mM phosphate is added to alleviate phosphate limitations and the Bis-Tris buffer concentration is increased to stabilize pH. By following radioactivity from [U-(14)C]-glucose, we find that glucose is primarily metabolized to the anaerobic products, acetate and lactate. The ability to use glucose as an energy source in cell-free reactions is important not only for inexpensive ATP generation during protein synthesis, but also as an example of how complex biological systems can be understood and exploited through cell-free biology.

    View details for DOI 10.1002/bit.20449

    View details for Web of Science ID 000229417600009

    View details for PubMedID 15830344

  • Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system BIOTECHNOLOGY AND BIOENGINEERING Yang, J. H., Kanter, G., Voloshin, A., Michel-Reydellet, N., Velkeen, H., Levy, R., Swartz, J. R. 2005; 89 (5): 503-511

    Abstract

    The idiotype (Id)-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins are potential vaccines for immunotherapy of B-cell lymphoma. In this study, four vaccine candidates were constructed by fusing murine GM-CSF to the amino- or carboxy-terminus of the 38C13 murine B-lymphocyte Id scFv with two different arrangements of the variable regions of the heavy chain and light chain (VL-VH and VH-VL). scFv (VH-VL) and GM-CSF/scFv fusion proteins were expressed in an Escherichia coli cell-free protein synthesis system. In order to promote disulfide bond formation during cell-free expression, cell extract was pretreated with iodoacetamide (IAM), and a sulfhydryl redox buffer composed of oxidized and reduced glutathione was added. The E. coli periplasmic disulfide isomerase, DsbC, was also added to rearrange incorrectly formed disulfide linkages. The 38C13 B-lymphocyte Id scFv was expressed with 30% of its soluble yield in active form (43 microg/ml) when tested with an anti-idiotypic mAb, S1C5, as the capture antibody in radioimmunoassay. It was found that the amino-terminal GM-CSF fusion proteins, GM-VL-VH and GM-VH-VL, showed much higher activity than the carboxy-terminal GM-CSF fusion proteins, VL-VH-GM and VH-VL-GM, in stimulating the cell proliferation of a GM-CSF-dependent cell line, NFS-60. Between the two amino-terminal GM-CSF fusion proteins, GM-VL-VH showed a higher total and soluble yield than GM-VH-VL.

    View details for Web of Science ID 000227171700002

    View details for PubMedID 15669088

  • Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis BIOTECHNOLOGY PROGRESS Liu, D. V., Zawada, J. F., Swartz, J. R. 2005; 21 (2): 460-465

    Abstract

    Escherichia coli extracts activate cell-free protein synthesis systems by providing the catalysts for translation and other supporting reactions. Recent results suggest that high-density fermentations can be used to provide the source cells, but the subsequent cell extract preparation procedure requires multiple centrifugation and dialysis steps as well as an expensive runoff reaction. In the work reported here, the extract preparation protocol duration was reduced by nearly 50% by significantly shortening several steps. In addition, by optimizing the runoff incubation, overall reagent costs were reduced by 70%. Nonetheless, extracts produced from the shorter, less expensive procedure were equally active. Crucial steps were further examined to indicate minimal ribosome loss during the standard 30,000g centrifugations. Furthermore, sucrose density centrifugation analysis indicated that although an incubation step significantly activates the extract, ribosome/polysome dissociation is not required. These insights suggest that consistent cell extract can be produced more quickly and with considerably less expense for large-scale cell-free protein production, especially when combined with high-density fermentation protocols.

    View details for DOI 10.1021/bp049789y

    View details for Web of Science ID 000228127200021

    View details for PubMedID 15801786

  • Maintaining rapid growth in moderate-density Escherichia coli fermentations BIOTECHNOLOGY AND BIOENGINEERING Zawada, J., Swartz, J. 2005; 89 (4): 407-415

    Abstract

    A novel feeding strategy that prolongs rapid growth rates for Escherichia coli fermentations to moderately high cell density is presented. High-density fermentations are a common and successful means of producing biological products. However, acetate accumulation can be a substantial problem in these procedures. To avoid this problem, many feeding strategies and host modifications have been developed, but all result in relatively low growth rates. If a faster growth rate could be maintained, the growth phase of the process would be shortened, leading to increased productivity. It is also possible that the subsequent specific production rate could be enhanced by growing the early culture at a faster rate. We have developed a procedure to enable rapid growth to a cell density of 20 g/L and have used cell-free protein synthesis to evaluate the relative potential of the resulting cells for producing recombinant proteins. The method uses glucose pulses and the duration of the dissolved oxygen response to calculate the appropriate glucose feed rate based on the glucose demand of the culture. Amino acids and vitamins were supplied in the medium to increase the growth rate. We were able to sustain a growth rate of 0.8/h up to 20 g/L dry cell weight without significant acetate accumulation. Analysis of amino acid consumption indicates that cell composition is an accurate predictor of amino acid demand for most amino acids. Cell-free protein synthesis was used to compare the protein production potential of the high-density cultures with that of cells grown in complex medium and harvested at low cell density and maximum growth rate. Protein production for the extract from the controlled, high-density fermentations was 950 mg/L compared with 860 mg/L for the low-density control. Therefore, the new control procedure has promising potential for developing rapid and productive industrial fermentations.

    View details for DOI 10.1002/bit.20369

    View details for Web of Science ID 000226701100005

    View details for PubMedID 15635610

  • Increasing PCR fragment stability and protein yields in a cell-free system with genetically modified Escherichia coli extracts JOURNAL OF MOLECULAR MICROBIOLOGY AND BIOTECHNOLOGY Michel-Reydellet, N., Woodrow, K., Swartz, J. 2005; 9 (1): 26-34

    Abstract

    Escherichia coli cell-free protein synthesis is a highly productive system that can be applied to high throughput expression from polymerase chain reaction (PCR) products in 96-well plates for proteomic studies as well as protein evolution. However, linear DNA instability appears to be a major limitation of the system. We modified the genome of the E. coli strain A19 by removing the endA gene encoding the endonuclease I and replacing the recCBD operon (in which recD encodes the exonuclease V) by the lambda phage recombination system. Using the cell extract from this new strain increased the stability of PCR products amplified from a plasmid containing the cat gene. This resulted in CAT (chloramphenicol acetyltransferase) production from PCR products comparable to that from plasmids (500-600 microg/ml) in a batch reaction. We show that cell-free protein synthesis reactions using PCR products amplified from genomic DNA and extended with the T7 promoter and the T7 terminator give the same high yields of proteins (550 microg/ml) in 96-well plates. With this system, it was possible to rapidly express a range of cytoplasmic and periplasmic proteins.

    View details for DOI 10.1159/000088143

    View details for Web of Science ID 000232991800003

    View details for PubMedID 16254443

  • Expression of active murine granulocyte-macrophage colony-stimulating factor in an Escherichia coli cell-free system BIOTECHNOLOGY PROGRESS Yang, J. H., Kanter, G., Voloshin, A., Levy, R., Swartz, J. R. 2004; 20 (6): 1689-1696

    Abstract

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM-CSF in an E. coli cell-free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form. However, the GM-CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM-CSF was produced in the cell-free reaction. Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.

    View details for DOI 10.1021/bp034350b

    View details for Web of Science ID 000225558200010

    View details for PubMedID 15575700

  • Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm BIOTECHNOLOGY AND BIOENGINEERING Jewett, M. C., Swartz, J. R. 2004; 87 (4): 465-472

    Abstract

    Cytoplasmic mimicry has recently led to the development of a novel method for cell-free protein synthesis called the "Cytomim" system. In vitro translation with this new system produced more than a 5-fold yield increase of chloramphenicol acetyl transferase (CAT) relative to a conventional method using pyruvate as an energy substrate. Factors responsible for activating enhanced protein yields, and causes leading to protein synthesis termination have been assessed in this new system. Enhanced yields were caused by the combination of three changes: growing the extract source cells on 2x YTPG media versus 2x YT, replacing polyethylene glycol with spermidine and putrescine, and reducing the magnesium concentration from conventional levels. Cessation of protein synthesis was primarily caused by depletion of cysteine, serine, CTP, and UTP. Substrate replenishment of consumed amino acids, CTP, and UTP extended the duration of protein synthesis to 24 h in fed-batch operation and produced 1.2 mg/mL of CAT. By also adding more T7 RNA polymerase and plasmid DNA, yields were further improved to 1.4 mg/mL of CAT. These results underscore the critical role that nucleotides play in the combined transcription-translation reaction and highlight the importance of understanding metabolic processes influencing substrate depletion.

    View details for DOI 10.1002/bit.20139

    View details for Web of Science ID 000223072500004

    View details for PubMedID 15286983

  • Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome METABOLIC ENGINEERING Michel-Reydellet, N., Calhoun, K., Swartz, J. 2004; 6 (3): 197-203

    Abstract

    Cell-free biology provides a unique opportunity to assess and to manipulate microbial systems by inverse metabolic engineering. We have applied this approach to amino acid metabolism, one of the systems in cell-free biology that limits protein synthesis reactions. Four amino acids (arginine, tryptophan, serine and cysteine) are depleted during a 3-h batch cell-free protein synthesis reaction under various conditions. By modifying the genome of the Escherichia coli strain used to make the cell extract, we see significant stabilization of arginine, tryptophan and serine. Cysteine, however, continues to be degraded. Cell-free protein synthesis with the modified cell extract produces increased yields of the cysteine-free protein Outer Membrane Protein T (OmpT).

    View details for DOI 10.1016/j.ymben.2004.01.003

    View details for Web of Science ID 000222938200004

    View details for PubMedID 15256209

  • Enhancing multiple disulfide bonded protein folding in a cell-free system BIOTECHNOLOGY AND BIOENGINEERING Yin, G., Swartz, J. R. 2004; 86 (2): 188-195

    Abstract

    A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell-free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli-based cell-free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30 degrees C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 microg/mL of bioactive PA in a simple 3-h batch reaction.

    View details for Web of Science ID 000220503000008

    View details for PubMedID 15052638

  • Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis BIOTECHNOLOGY AND BIOENGINEERING Jewett, M. C., Swartz, J. R. 2004; 86 (1): 19-26

    Abstract

    Cell-free translation systems generally utilize high-energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 microg/mL of protein. By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds.

    View details for DOI 10.1002/bit.20026

    View details for Web of Science ID 000220196000003

    View details for PubMedID 15007837

  • Cell-free production of active E-coli thioredoxin reductase and glutathione reductase FEBS LETTERS Knapp, K. G., Swartz, J. R. 2004; 559 (1-3): 66-70

    Abstract

    Escherichia coli thioredoxin reductase (TR) and glutathione reductase (GR) are dimeric proteins that require a flavin adenine dinucleotide (FAD) cofactor for activity. A cell-free protein synthesis (CFPS) reaction supplemented with FAD was used to produce TR at 760 microg/ml with 89% of the protein being soluble. GR accumulated to 521 microg/ml in a cell-free reaction with 71% solubility. The TR produced was fully active with a specific activity of 1390 min(-1). The GR had a specific activity of 139 U/mg, which is significantly more active than reported for GR purified from cells. The specific activity for both TR and GR decreased without FAD supplementation. This research demonstrates that CFPS can be used to produce enzymes that are multimeric and require a cofactor.

    View details for DOI 10.1016/S0014-5793(04)00025-0

    View details for Web of Science ID 000188970100012

    View details for PubMedID 14960309

  • Genetic analysis of disulfide isomerization in Escherichia coli: Expression of DsbC is modulated by RNase e-dependent mRNA processing JOURNAL OF BACTERIOLOGY Zhan, X. M., Gao, J. J., Jain, C. T., Cieslewicz, M. J., Swartz, J. R., Georgiou, G. 2004; 186 (3): 654-660

    Abstract

    We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity. The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell. Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain. Following chemical mutagenesis, a total of eight E. coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated. This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC. In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product. Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci. The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37 degrees C in wild-type cells but was two- to threefold longer in some of the stronger mutants. These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression.

    View details for DOI 10.1128/JB.186.3.654-660.2004

    View details for Web of Science ID 000188371600008

    View details for PubMedID 14729690

  • Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli BIOTECHNOLOGY AND BIOENGINEERING Kim, D. M., Swartz, J. R. 2004; 85 (2): 122-129

    Abstract

    In this report, we demonstrate that a complex mammalian protein containing multiple disulfide bonds is successfully expressed in an E.coli-based cell-free protein synthesis system. Initially, disulfide-reducing activities in the cell extract prevented the formation of disulfide bonds. However, a simple pretreatment of the cell extract with iodoacetamide abolished the reducing activity. This extract was still active for protein synthesis even under oxidizing conditions. The use of a glutathione redox buffer coupled with the DsbC disulfide isomerase and pH optimization produced 40 microg/mL of active urokinase protease in a simple batch reaction. This result not only demonstrates efficient production of complex proteins, it also emphasizes the control and flexibility offered by the cell-free approach.

    View details for DOI 10.1002/bit.10865

    View details for Web of Science ID 000187634700002

    View details for PubMedID 14704994

  • Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis BIOTECHNOLOGY PROGRESS Jewett, M. C., Swartz, J. R. 2004; 20 (1): 102-109

    Abstract

    Two strategies for ATP regeneration during cell-free protein synthesis were applied to the large-scale production and single-column purification of active chloramphenicol acetyl transferase (CAT). Fed-batch reactions were performed on a 5-10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT in a 5 mL reaction over the course of 5 h. The PANOx system produced 261 +/- 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatography adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed-batch cell-free protein synthesis reaction.

    View details for DOI 10.1021/bp0341693

    View details for Web of Science ID 000188861300014

    View details for PubMedID 14763830

  • Cell-free protein synthesis with prokaryotic combined transcription-translation. Methods in molecular biology (Clifton, N.J.) Swartz, J. R., Jewett, M. C., Woodrow, K. A. 2004; 267: 169-182

    Abstract

    Cell-free biology exploits and studies complex biological processes in a controlled environment without intact cells. One model system is prokaryotic cell-free protein synthesis. This technology offers an attractive and convenient approach to produce properly folded recombinant DNA (rDNA) proteins on a laboratory scale, screen PCR fragment libraries in a high-throughput format, express pharmaceutical proteins, incorporate labeled or unnatural amino acids into proteins, and activate microbial physiology to allow for investigation of biological systems. We describe the preparation of materials necessary for the expression, quantification, and purification of rDNA proteins from active Escherichia coli extracts.

    View details for PubMedID 15269424

  • Affinity purification of lipid vesicles BIOTECHNOLOGY PROGRESS Peker, B., Wuu, J. J., Swartz, J. R. 2004; 20 (1): 262-268

    Abstract

    We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.

    View details for DOI 10.1021/bp034182n

    View details for Web of Science ID 000188861300035

    View details for PubMedID 14763851

  • Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis BIOTECHNOLOGY AND BIOENGINEERING Kim, D. M., Swartz, J. R. 2001; 74 (4): 309-316

    Abstract

    A new approach for adenosine triphosphate (ATP) regeneration in a cell-free protein synthesis system is described. We first show that pyruvate can be used as a secondary energy source to replace or supplement the conventional secondary energy source, phosphoenol pyruvate (PEP). We also report that glucose-6-phosphate, an earlier intermediate of the glycolytic pathway, can be used for ATP regeneration. These new methods provide more stable maintenance of ATP concentration during protein synthesis. Because pyruvate and glucose-6-phosphate are the first and last intermediates of the glycolytic pathway, respectively, the results also suggest the possibility of using any glycolytic intermediate, or even glucose, for ATP regeneration in a cell-free protein synthesis system. As a result, the methods described provide cell-free protein synthesis with greater flexibility and cost efficiency.

    View details for Web of Science ID 000169835000005

    View details for PubMedID 11410855

  • A PURE approach to constructive biology NATURE BIOTECHNOLOGY Swartz, J. 2001; 19 (8): 732-733

    View details for Web of Science ID 000170188600020

    View details for PubMedID 11479561

  • Advances in Escherichia coli production of therapeutic proteins CURRENT OPINION IN BIOTECHNOLOGY Swartz, J. R. 2001; 12 (2): 195-201

    Abstract

    Escherichia coli offers a means for the rapid and economical production of recombinant proteins. These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically sensitive products as insulin and bovine growth hormone. Although significant progress has been made in transcription, translation and secretion, one of the major challenges is obtaining the product in a soluble and bioactive form. Recent progress in oxidative cytoplasmic folding and cell-free protein synthesis offers attractive alternatives to standard expression methods.

    View details for Web of Science ID 000167915900015

    View details for PubMedID 11287237

  • Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli JOURNAL OF BACTERIOLOGY Bessette, P. H., Qiu, J., Bardwell, J. C., Swartz, J. R., Georgiou, G. 2001; 183 (3): 980-988

    Abstract

    We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.

    View details for Web of Science ID 000166459500022

    View details for PubMedID 11208797

  • Oxalate improves protein synthesis by enhancing ATP supply in a cell-free system derived from Escherichia coli BIOTECHNOLOGY LETTERS Kim, D. M., Swartz, J. R. 2000; 22 (19): 1537-1542
  • Prolonging cell-free protein synthesis by selective reagent additions BIOTECHNOLOGY PROGRESS Kim, D. M., Swartz, J. R. 2000; 16 (3): 385-390

    Abstract

    Factors causing the early cessation of protein synthesis have been studied in a cell-free system from Escherichia coli. We discovered that phosphoenol pyruvate (PEP), the secondary energy source for ATP regeneration, and several amino acids are rapidly degraded during the cell-free protein synthesis reaction. The degradation of such compounds takes place even in the absence of protein synthesis. This degradation severely reduces the capacity for protein synthesis. The lost potency was completely recovered when the reaction mixture was supplied with additional PEP and amino acids. Of the 20 amino acids, only arginine, cysteine, and tryptophan were required to restore system activity. Through repeated additions of PEP, arginine, cysteine,and tryptophan, the duration of protein synthesis was greatly extended. In this fed-batch reaction, after a 2 h incubation, the level of cell-free synthesized chloramphenicol acetyl transferase (CAT) reached 350 microg/mL, which is 3.5 times the yield of the batch reaction. Addition of fresh magnesium further extended the protein synthesis. As a result, through coordinated additions of PEP, arginine, cysteine, tryptophan, and magnesium, the final concentration of cell-free synthesized CAT increased more than 4-fold compared to a batch reaction. SDS-PAGE analysis of such a fed-batch reaction produced an obvious band of CAT upon Coomassie Blue staining.

    View details for Web of Science ID 000087483800013

    View details for PubMedID 10835240

  • Prolonging cell-free protein synthesis with a novel ATP regeneration system BIOTECHNOLOGY AND BIOENGINEERING Kim, D. M., Swartz, J. R. 1999; 66 (3): 180-188

    Abstract

    A new approach for the regeneration of adenosine triphosphate (ATP) during cell-free protein synthesis was developed to prolong the synthesis and also to avoid the accumulation of inorganic phosphate. This approach was demonstrated in a batch system derived from Escherichia coli. Contrary to the conventional methods in which exogenous energy sources contain high-energy phosphate bonds, the new system was designed to generate continuously the required high-energy phosphate bonds within the reaction mixture, thereby recycling the phosphate released during protein synthesis. If allowed to accumulate, phosphate inhibits protein synthesis, most likely by reducing the concentration of free magnesium ion. Pediococcus sp. pyruvate oxidase, when introduced in the reaction mixture along with thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), catalyzed the generation of acetyl phosphate from pyruvate and inorganic phosphate. Acetyl kinase, already present with sufficient activity in Escherichia coli S30 extract, then catalyzed the regeneration of ATP. Oxygen is required for the generation of acetyl phosphate and the H(2)O(2) produced as a byproduct is sufficiently degraded by endogenous catalase activity. Through the continuous supply of chemical energy, and also through the prevention of inorganic phosphate accumulation, the duration of protein synthesis is extended up to 2 h. Protein accumulation levels also increase. The synthesis of human lymphotoxin receives greater benefit than than that of chloramphenicol acetyl transferase, because the former is more sensitive to phosphate inhibition. Finally, through repeated addition of pyruvate and amino acids during the reaction period, protein synthesis continued for 6 h in the new system, resulting in a final yield of 0.7 mg/mL.

    View details for Web of Science ID 000084141100006

    View details for PubMedID 10577472

Conference Proceedings


  • Binding of a cationic protein to the cell surface is insufficient for cellular uptake and bioactivity: Arginine-rich sequences are necessary Yang, W. C., Lee, J., Albayrak, C., Cooke, J. P., Swartz, J. R. AMER CHEMICAL SOC. 2011
  • High-yield production of transducible transcription factors for non-viral modulation of gene expression Yang, W. C., Welsh, J. P., Cooke, J. P., Swartz, J. R. AMER CHEMICAL SOC. 2010
  • Cell-free incorporation of non-natural amino acids in proteins enables the production of complex protein bioconjugates Patel, K. G., Swartz, J. R. AMER CHEMICAL SOC. 2010
  • Effective site-specific incorporation of non-natural amino acids via simultaneous cell-free synthesis of the orthogonal tRNA and the product protein Albayrak, C., Swartz, J. R. AMER CHEMICAL SOC. 2010
  • Novel Anti-CD19/Idiotype Bispecific Diabody Vaccine for B-Cell Lymphoma Ng, P. P., Jia, M., Virrueta, A., Patel, K., Swartz, J. R., Levy, S., Levy, R. AMER SOC HEMATOLOGY. 2009: 1062-1062
  • BIOT 10-Cell-free protein synthesis of complex proteins and protein assemblies containing posttranslational modification Goerke, A. R., Wuu, J. J., Ebina, W., Bundy, B. C., Swartz, J. R. AMER CHEMICAL SOC. 2008
  • BIOT 496-Activating and evolving hydrogenases for solar hydrogen production Swartz, J. R., Stapleton, J. A., Kuchenreuther, J. M., Smith, P. AMER CHEMICAL SOC. 2008
  • BIOT 143-Development of in vivo and in vitro systems for studying the expression and activation of [FeFe] hydrogenases and their required maturases Kuchenreuther, J. M., Boyer, M. E., Stapleton, J. A., Swartz, J. R. AMER CHEMICAL SOC. 2008
  • BIOT 482-Enhancing production of complex mammalian proteins using E-coli based cell-free protein synthesis Welsh, J. P., Swartz, J. R., Bonomo, J. AMER CHEMICAL SOC. 2008
  • BIOT 345-High yields of complex proteins with site-specific posttranslational modification using cell-free protein synthesis Goerke, A. R., Wuu, J. J., Ebina, W., Swartz, J. R. AMER CHEMICAL SOC. 2007
  • BIOT 106-"Seeing the light" with cell-free protein synthesis Goerke, A. R., Loening, A. M., Gambhir, S., Swartz, J. R. AMER CHEMICAL SOC. 2007
  • BIOT 364-Expressing high yields of membrane proteins using cell-free synthesis Wuu, J. J., Swartz, J. R. AMER CHEMICAL SOC. 2006
  • BIOT 70-Avoiding mass transport limitations in hydrophobic biotransformations by efficient cell localization Yancey, D. D., Robertson, C. R., Swartz, J. AMER CHEMICAL SOC. 2006
  • BIOT 396-Escherichia coli-based cell-free protein synthesis of empty viral capsids Bundy, B. C., Swartz, J. R. AMER CHEMICAL SOC. 2006
  • BIOT 388-Extending cell-free protein synthesis to complex targets: Expression and maturation of FeFe-hydrogenases Boyer, M. E., Stapleton, J. A., Kuchenreuther, J. M., Wang, C., Swartz, J. R. AMER CHEMICAL SOC. 2006
  • BIOT 4-Developing a cell-free protein synthesis platform for producing proteins requiring disulfide bonds Goerke, A. R., Knapp, K. G., Swartz, J. R. AMER CHEMICAL SOC. 2006
  • BIOT 22-Directed evolution of oxygen-tolerant hydrogenases for photobiological hydrogen production Stapleton, J. A., Boyer, M. E., Swartz, J. R. AMER CHEMICAL SOC. 2006
  • Developing cell-free biology for industrial applications Swartz, J. SPRINGER HEIDELBERG. 2006: 476-485

    Abstract

    Although cell-free protein synthesis has been practiced for decades as a research tool, only recently have advances suggested its feasibility for commercial protein production. This focused review, based on the 2005 Amgen Award lecture, summarizes the relevant progress from the Swartz laboratory. When our program began, projected costs were much too high, proteins with disulfide bonds could not be folded effectively, and no economical scale-up technologies were available. By focusing on basic biochemical reactions and by controlling cell-free metabolism, these limitations have been methodically addressed. Amino acid supply has been stabilized and central metabolism activated to dramatically reduce substrate costs. Control of the sulfhydral redox potential has been gained and a robust disulfide isomerase added to facilitate oxidative protein folding. Finally, simple scale-up technologies have been developed. These advances not only suggest production feasibility for pharmaceutical proteins, they also provide enabling technology for producing patient-specific vaccines, for evolving new enzymes to enable biological hydrogen production from sunlight, and for developing new and highly effective water filters. Although many challenges remain, this newly expanded ability to activate and control protein production holds much promise for both research and commercial applications.

    View details for DOI 10.1007/s10295-006-0127-y

    View details for Web of Science ID 000239443800002

    View details for PubMedID 16761165

  • Maintaining stable amino acid concentrations during cell-free protein synthesis. Calhoun, K. A., Michel-Reydellet, N., Swartz, J. R. AMER CHEMICAL SOC. 2005: U229-U229
  • Metabolic modeling of cell-free protein synthesis reactions. Calhoun, K. A., Varner, J., Jewett, M. C., Swartz, J. R. AMER CHEMICAL SOC. 2005: U194-U194
  • Functional genomic analysis using in vitro protein expression and folding. Woodrow, K. A., Swartz, J. R. AMER CHEMICAL SOC. 2005: U190-U191
  • Cell-free synthesis enables patient-specific vaccine production. Swartz, J. R., Levy, R., Kanter, G., Yang, J. H., Voloshin, A. AMER CHEMICAL SOC. 2005: U202-U203
  • Economic cell-free protein synthesis system energized with glucose metabolism and NMPS. Calhoun, K. A., Swartz, J. R. AMER CHEMICAL SOC. 2005: U180-U180
  • A novel method for producing custom-made idiotype vaccines for lymphoma immunotherapy using a cell-free expression system. Kanter, G., Yang, J. H., Voloshin, A., Swartz, J. R., Levy, R. AMER SOC HEMATOLOGY. 2004: 395A-395A
  • Protein expression and engineering for structural studies. Maynard, J., Garcia, K. C. AMER CHEMICAL SOC. 2004: U131-U131
  • Cell-free protein synthesis platform: Ideal system for unnatural amino acid incorporation. Schulte, J., Swartz, J. R. AMER CHEMICAL SOC. 2004: U129-U129
  • Toward protein glycosylation through post-translational modification from a cell-free protein synthesis system. Schulte, J., Swartz, J. R. AMER CHEMICAL SOC. 2004: U219-U219
  • Using cell-free biology to study systems biology. Swartz, J. R., Calhoun, K. A., Jewett, M. C. AMER CHEMICAL SOC. 2004: U255-U255
  • Rapid expression of vaccine proteins for B cell lymphoma in a cell-free system. Yang, J. H., Kanter, G., Voloshin, A., Levy, R., Swartz, J. AMER CHEMICAL SOC. 2004: U137-U137
  • Rapid expression of vaccine proteins for B cell lymphoma in a cell-free system. Yang, J. H., Kanter, G., Voloshin, A., Levy, R., Swartz, J. AMER CHEMICAL SOC. 2004: U135-U135
  • In vitro system for simultaneous expression and maturation of iron-sulfur proteins. Boyer, M. E., Wang, C. W., Spormann, A. M., Swartz, J. AMER CHEMICAL SOC. 2004: U225-U225
  • Accelerating product and process development by using cell-free protein synthesis. Swartz, J. R., Michelle-Reydellet, N., Knapp, K., Yin, G., Yang, J. H. AMER CHEMICAL SOC. 2003: U190-U190
  • Carbon flux analysis of metabolic reactions in cell-free protein synthesis. Calhoun, K. A., Swartz, J. R. AMER CHEMICAL SOC. 2003: U214-U214
  • Cell-free protein synthesis utilizing unnatural amino acids Schulte, J., Swartz, J. R. AMER CHEMICAL SOC. 2003: U186-U186
  • Mimicking the cytoplasmic environment of Escherichia coli with cell-free protein synthesis activates oxidative phosphorylation Jewett, M. C., Swartz, J. R. AMER CHEMICAL SOC. 2003: U185-U185
  • Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis. Liu, D. V., Zawada, J. F., Swartz, J. R. AMER CHEMICAL SOC. 2003: U230-U230
  • Isolation and genetic analysis of mutation that increase disulfide isomerization activity in E. coli Zhan, X. M., Swartz, J. R., Georgiou, G. AMER CHEMICAL SOC. 2002: U231-U231
  • Effects of genotype and growth conditions on cell-free protein synthesis systems. Zawada, J., Richter, B., Huang, E., Lodes, E., Shah, A., Swartz, J. R. AMER CHEMICAL SOC. 2002: U213-U213
  • Engineering cell-free protein synthesis for efficient protein folding. Swartz, J. R., Yin, G., Yang, J. H., Voloshin, A., Jewett, M. C., Woodrow-Mumford, K. AMER CHEMICAL SOC. 2002: U230-U230
  • Construction of a protein crystallographic calculator. Shanbhag, P. P., Gonzalez, A. AMER CHEMICAL SOC. 2002: U220-U220
  • Metabolic oscillations in an E-coli fermentation. Andersen, D. C., Swartz, J. R., Ryll, T., Lin, N., Snedecor, B. AMER CHEMICAL SOC. 2001: U116-U117
  • Synthesis of mammalian proteins with an E.coli cell-free system. Swartz, J. R., Kim, D. M., Michel-Reydellet, N., Zawada, J., Jewett, M., Schulte, J. AMER CHEMICAL SOC. 2001: U108-U108
  • Bioenergetics in cell-free protein synthesis systems. Zawada, J. F., Kim, D. M., Swartz, J. R. AMER CHEMICAL SOC. 2000: U173-U173
  • Designing cell-free protein synthesis for efficient protein folding. Kim, D. M., Swartz, J. R. AMER CHEMICAL SOC. 2000: U190-U190
  • Cell-free protein expression: Designing for efficiency and low cost. Swartz, J. R., Kim, D. M., Zawada, J., Michel-Reydellet, N. AMER CHEMICAL SOC. 2000: U166-U166
  • Cell-free protein synthesis: New analyses; new efficiencies. Swartz, J. R., Kim, D. M. AMER CHEMICAL SOC. 1999: U182-U182