RT Journal Article SR Electronic T1 Periplasmic Expression of SpyTagged Antibody Fragments Enables Rapid Modular Antibody Assembly JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.09.18.302950 DO 10.1101/2020.09.18.302950 A1 Hentrich, Christian A1 Kellmann, Sarah-Jane A1 Putyrski, Mateusz A1 Cavada, Manuel A1 Hanuschka, Hanh A1 Knappik, Achim A1 Ylera, Francisco YR 2020 UL http://biorxiv.org/content/early/2020/09/20/2020.09.18.302950.abstract AB Antibodies 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.Competing Interest StatementAll authors are employees of Bio-Rad AbD Serotec GmbH. Bio-Rad Laboratories, Inc. filed patent applications on technologies described herein, on which A.K., C.H. and F.Y. are listed as inventors.