Periplasmic Expression of SpyTagged Antibody Fragments Enables Rapid Modular Antibody Assembly

Abstract

AbstractAntibodies are essential tools in research and diagnostics. While antibody fragments can be rapidly produced in Escherichia coli, full-length antibodies with an Fc region or antibodies modified with probes are time and labor intensive in production.SpyTag/SpyCatcher protein ligation technology could covalently attach such functionalities to antibody fragments equipped with a SpyTag. However, we found that the necessarily periplasmic expression of such antibody fragments in E. coli led to rapid cleavage of the SpyTag by proteases.Here we show how this cleavage can be prevented, making the SpyTag technology accessible for E. coli produced antibodies. We demonstrate a modular toolbox for rapid creation of synthetic IgGs, oligomerized antibodies, and antibodies with different tags or enzymatic functionalities and measure their performance in a variety of immunoassays. Furthermore, we demonstrate surface immobilization, high-throughput screening of antibody libraries, and rapid prototyping of antibodies based on modular antibody assembly.