Sortase as a Tool in Biotechnology and Medicine

We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics.

We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.

We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells

Institutions, Innovation, and Grassroots Change: Alternatives to Transnational Governance in the Global South

Transnational governance has been advanced as a viable option for regulating commodities produced in emerging economies—where incapable or unwilling states may undersupply institutions requisite for overseeing supply chains consistent with the quality, safety, environmental, or social standards demanded by the global marketplace. Producers from these jurisdictions, otherwise left with few venues for securing market access and price premiums, ostensibly benefit from whatever pathways transnational actors offer to minimize barriers to entry—including voluntary certification for compliance with a panoply of public and private rules, such as those promulgated by NGOs like the Fair Trade Federation or multinational retailers like Wal-Mart. Yet, such transnational “sustainability” governance may neither be effective nor desirable. Regulatory schemes, like third-party certification, often privilege the interests of primary architects and beneficiaries—private business associations, governments, NGOs, and consumers in the global North—over regulatory targets—producers in the global South. Rather than engaging with the international marketplace via imported and externally-driven schemes, some producer groups are instead challenging existing rules and innovating homegrown institutions. These alternatives to commercialization adopt some institutional characteristics of their transnational counterparts yet deliver benefits in a manner more aligned with the needs of producers. Drawing on original empirical cases from Nicaragua and Mexico, this dissertation examines the role of domestic institutional alternatives to transnational governance in enhancing market access, environmental quality and rural livelihoods within producer communities. Unlike the more technocratic and expert-driven approaches characteristic of mainstream governance efforts, these local regulatory institutions build upon the social capital, indigenous identity, “ancestral” knowledge, and human assets of producer communities as new sources of power and legitimacy in governing agricultural commodities.