SpyDisplay: A versatile phage display selection system using SpyTag/SpyCatcher technology

ABSTRACT

Phage display is an established method for the in vitro selection of recombinant antibodies and other proteins or peptides from gene libraries. Here we describe SpyDisplay, a phage display method in which the display is achieved via SpyTag/SpyCatcher protein ligation instead of genetically fusing the displayed protein to a phage coat protein. In our implementation, SpyTagged antibody antigen-binding fragments (Fabs) are displayed via protein ligation on filamentous phages carrying SpyCatcher fused to the pIII coat protein. A library of genes encoding Fab antibodies was cloned in an expression vector containing an f1 replication origin, and SpyCatcher-pIII was separately expressed from a genomic locus in engineered E. coli. We demonstrate the functional, covalent display of Fab on phage, and rapidly isolate specific high-affinity clones via phage panning, confirming the robustness of this selection system. SpyTagged Fabs, the direct outcome of the panning campaign, are compatible with modular antibody assembly using prefabricated SpyCatcher modules and can be directly tested in diverse assays. Furthermore, SpyDisplay streamlines additional applications that have traditionally been challenging for phage display: we show that it can be applied to N-terminal display of the protein of interest and it enables display of cytoplasmically folding proteins exported to periplasm via the TAT pathway.

KEYWORDS:

• SpyTag/SpyCatcher
• antibody phage display
• SpyDisplay
• Fab antibody library

Abbreviations

CDR: complementarity-determining region; cfu: colony-forming unit; Fab: antigen binding fragment of an antibody; FRT: flippase recognition target; HRP: horseradish peroxidase; IgG: immunoglobulin G; IPTG: isopropyl β-D-1-thiogalactopyranoside; κ: kappa light chain of an antibody; λ: lambda light chain of an antibody; MBP: maltose-binding protein; mGFP: monomeric green fluorescent protein; pIII: filamentous phage protein III; TAT: twin arginine translocase; TEV: tobacco etch virus; ELISA: enzyme-linked immunosorbent assay; BLI: biolayer interferometry; PBS: phosphate-buffered saline; TBS: tris-buffered saline; BSA: bovine serum albumin; PEG: polyethylene glycol; scFv: single-chain variable fragment; SD: standard deviation

Acknowledgments

We thank our colleagues in the Fab production and assay teams for antibody purification and quality control and our colleagues in lab support for production of the library phages. We thank Melissa Wich for critical reading of the manuscript.

Contributions

S.-J.K., H.H., M.C., C.H., and M.P. performed experiments. S.-J.K., C.H., M.P., and F.Y. designed experiments and analyzed the data. A.K. and F.Y. conceived the project. C.H., S.-J.K., M.P., A.K., and F.Y. wrote the manuscript.

Disclosure statement

All authors are employees of Bio-Rad AbD Serotec GmbH. Bio-Rad Laboratories, Inc. filed patent applications on technologies described herein, on which F.Y. is listed as inventor.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2177978

Additional information

Funding

The authors reported there is no funding associated with the work featured in this article.