All Publications

  • Design of a mucin-selective protease for targeted degradation of cancer-associated mucins. Nature biotechnology Pedram, K., Shon, D. J., Tender, G. S., Mantuano, N. R., Northey, J. J., Metcalf, K. J., Wisnovsky, S. P., Riley, N. M., Forcina, G. C., Malaker, S. A., Kuo, A., George, B. M., Miller, C. L., Casey, K. M., Vilches-Moure, J. G., Ferracane, M. J., Weaver, V. M., Läubli, H., Bertozzi, C. R. 2023


    Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

    View details for DOI 10.1038/s41587-023-01840-6

    View details for PubMedID 37537499

    View details for PubMedCentralID 6031612

  • Classification, structural biology, and applications of mucin domain-targeting proteases. The Biochemical journal Shon, D. J., Kuo, A., Ferracane, M. J., Malaker, S. A. 2021; 478 (8): 1585–1603


    Epithelial surfaces throughout the body are coated by mucins, a class of proteins carrying domains characterized by a high density of O-glycosylated serine and threonine residues. The resulting mucosal layers form crucial host-microbe interfaces that prevent the translocation of microbes while also selecting for distinct bacteria via the presented glycan repertoire. The intricate interplay between mucus production and breakdown thus determines the composition of the microbiota maintained within these mucosal environments, which can have a large influence on the host during both homeostasis and disease. Most research to date on mucus breakdown has focused on glycosidases that trim glycan structures to release monosaccharides as a source of nutrients. More recent work has uncovered the existence of mucin-type O-glycosylation-dependent proteases that are secreted by pathogens, commensals, and mutualists to facilitate mucosal colonization and penetration. Additionally, immunoglobulin A (IgA) proteases promote bacterial colonization in the presence of neutralizing secretory IgA through selective cleavage of the heavily O-glycosylated hinge region. In this review, we summarize families of O-glycoproteases and IgA proteases, discuss known structural features, and review applications of these enzymes to glycobiology.

    View details for DOI 10.1042/BCJ20200607

    View details for PubMedID 33909028

  • Development of a general defined medium for Pichia pastoris BIOTECHNOLOGY AND BIOENGINEERING Matthews, C. B., Kuo, A., Love, K. R., Love, J. 2018; 115 (1): 103–13


    Pichia pastoris is widely used as a host for recombinant protein production. More than 500 proteins have been expressed in the organism at a variety of cultivation scales, from small shake flasks to large bioreactors. Large-scale fermentation strategies typically employ chemically defined growth medium because of its greater batch-to-batch consistency and in many cases, lower costs compared to complex medium. For biopharmaceuticals, defined growth medium may also simplify downstream purification and regulatory documentation. Standard formulations of defined media for P. pastoris are minimal ones that lack the metabolic intermediates provided by complex components such as peptone and yeast extract. As a result, growth rates and per-cell productivities are significantly lower than in complex medium. We have designed a rich defined medium (RDM) for Pichia pastoris by systematically evaluating nutrients of increasing complexity and identifying those that are most critical for growth. We have also employed transcriptomics to gain deeper insights into the underlying metabolic processes and inform our media design. We have demonstrated that using RDM for expression of three heterologous proteins yields titers comparable to, or higher than, those in standard complex medium. RDM improves productivity of P. pastoris fermentations and its development demonstrates the usefulness of transcriptomics to accelerate process development for new molecules.

    View details for DOI 10.1002/bit.26440

    View details for Web of Science ID 000416114400009

    View details for PubMedID 28865117

  • Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms BIOTECHNOLOGY AND BIOENGINEERING Matthews, C. B., Wright, C., Kuo, A., Colant, N., Westoby, M., Love, J. 2017; 114 (11): 2432–44


    Antibodies are an important class of therapeutics and are predominantly produced in Chinese Hamster Ovary (CHO) cell lines. While this manufacturing platform is sufficiently productive to supply patient populations of currently approved therapies, it is unclear whether or not the current CHO platform can address two significant areas of need: affordable access to biologics for patients around the globe and production of unprecedented quantities needed for very large populations of patients. Novel approaches to recombinant protein production for therapeutic biologic products may be needed, and might be enabled by non-mammalian expression systems and recent advances in bioengineering. Eukaryotic microorganisms such as fungi, microalgae, and protozoa offer the potential to produce high-quality antibodies in large quantities. In this review, we lay out the current understanding of a wide range of species and evaluate based on theoretical considerations which are best poised to deliver a step change in cost of manufacturing and volumetric productivity within the next decade.Related article:

    View details for DOI 10.1002/bit.26378

    View details for Web of Science ID 000411699200002

    View details for PubMedID 28688187