Laura Guerrero

All Publications

• A rapid cell-free expression and screening platform for antibody discovery. Nature communications Hunt, A. C., Vögeli, B., Hassan, A. O., Guerrero, L., Kightlinger, W., Yoesep, D. J., Krüger, A., DeWinter, M., Diamond, M. S., Karim, A. S., Jewett, M. C. 2023; 14 (1): 3897

  Abstract

  Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly.

  View details for DOI 10.1038/s41467-023-38965-w

  View details for PubMedID 37400446

  View details for PubMedCentralID PMC10318062

• Point-of-Care Peptide Hormone Production Enabled by Cell-Free Protein Synthesis ACS SYNTHETIC BIOLOGY DeWinter, M. A., Thames, A., Guerrero, L., Kightlinger, W., Karim, A. S., Jewett, M. C. 2023; 12 (4): 1216-1226

  Abstract

  In resource-limited settings, it can be difficult to safely deliver sensitive biologic medicines to patients due to cold chain and infrastructure constraints. Point-of-care drug manufacturing could circumvent these challenges since medicines could be produced locally and used on-demand. Toward this vision, we combine cell-free protein synthesis (CFPS) and a 2-in-1 affinity purification and enzymatic cleavage scheme to develop a platform for point-of-care drug manufacturing. As a model, we use this platform to synthesize a panel of peptide hormones, an important class of medications that can be used to treat a wide variety of diseases including diabetes, osteoporosis, and growth disorders. With this approach, temperature-stable lyophilized CFPS reaction components can be rehydrated with DNA encoding a SUMOylated peptide hormone of interest when needed. Strep-Tactin affinity purification and on-bead SUMO protease cleavage yield peptide hormones in their native form that are recognized by ELISA antibodies and that can bind their respective receptors. With further development to ensure proper biologic activity and patient safety, we envision that this platform could be used to manufacture valuable peptide hormone drugs in a decentralized way.

  View details for DOI 10.1021/acssynbio.2c006801216

  View details for Web of Science ID 000975893300001

  View details for PubMedID 36940255

• Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds METABOLIC ENGINEERING COMMUNICATIONS Arvay, E., Biggs, B. W., Guerrero, L., Jiang, V., Tyo, K. 2021; 13: e00173

  Abstract

  Utilization of lignin, an abundant renewable resource, is limited by its heterogenous composition and complex structure. Biological valorization of lignin provides advantages over traditional chemical processing as it occurs at ambient temperature and pressure and does not use harsh chemicals. Furthermore, the ability to biologically funnel heterogenous substrates to products eliminates the need for costly downstream processing and separation of feedstocks. However, lack of relevant metabolic networks and low tolerance to degradation products of lignin limits the application of traditional engineered model organisms. To circumvent this obstacle, we employed Acinetobacter baylyi ADP1, which natively catabolizes lignin-derived aromatic substrates through the β-ketoadipate pathway, to produce mevalonate from lignin-derived compounds. We enabled expression of the mevalonate pathway in ADP1 and validated activity in the presence of multiple lignin-derived aromatic substrates. Furthermore, by knocking out wax ester synthesis and utilizing fed-batch cultivation, we improved mevalonate titers 7.5-fold to 1014 mg/L (6.8 mM). This work establishes a foundation and provides groundwork for future efforts to engineer improved production of mevalonate and derivatives from lignin-derived aromatics using ADP1.

  View details for DOI 10.1016/j.mec.2021.e00173

  View details for Web of Science ID 000905588400002

  View details for PubMedID 34430203

  View details for PubMedCentralID PMC8367835

• YTHDC1 mediates nuclear export of N-6 - methyladenosine methylated mRNAs ELIFE Roundtree, I. A., Luo, G., Zhang, Z., Wang, X., Zhou, T., Cui, Y., Sha, J., Huang, X., Guerrero, L., Xie, P., He, E., Shen, B., He, C. 2017; 6

  Abstract

  N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m6A-selective 'reader' proteins of the YTH family, which incorporate m6A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m6A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m6A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m6A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

  View details for DOI 10.7554/eLife.31311

  View details for Web of Science ID 000413185800001

  View details for PubMedID 28984244

  View details for PubMedCentralID PMC5648532