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 Christian Hentrich
A1 Sarah-Jane Kellmann
A1 Mateusz Putyrski
A1 Manuel Cavada
A1 Hanh Hanuschka
A1 Achim Knappik
A1 Francisco Ylera
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.