Cell Surface Display Yields Evolvable, Clickable Antibody Fragments
Abstract
Non-canonical amino acids (ncAAs) provide powerful tools for engineering the chemical and physical properties of proteins. However, introducing ncAAs into proteins can affect protein properties in unpredictable ways, thus necessitating screening efforts to identify mutants with desirable properties. In this work, we describe an Escherichia coli cell surface display platform for the directed evolution of clickable antibody fragments. This platform enabled isolation of antibody fragments with improved digoxigenin binding and modest affinity maturation in several different ncAA contexts. Azide-functionalized fragments exhibited improved binding kinetics relative to their methionine counterparts, facile chemical modification through azide–alkyne cycloaddition, and retention of binding properties after modification. The results described here suggest new possibilities for protein engineering, including modulation of molecular recognition events by ncAAs and direct screening of libraries of chemically modified proteins.
Additional Information
© 2014 Wiley-VCH Verlag GmbH & Co. Article first published online: 17 Jul 2014. We thank Mona Shahgholi for MALDI mass spectrometry assistance at the Caltech Chemistry Mass Spectrometry facility, Igor Antoshechkin and the Millard and Muriel Jacobs Genetics and Genomics Laboratory for assistance with high-throughput sequencing, Cynthia Shuman of GE Healthcare for Biacore kinetic assay guidance, Janek Szychowski and Alborz Mahdavi for materials, and Maren Buck for helpful comments on the manuscript. This work was supported by NIH grant R01 GM62523. J.A.V. was supported in part by a National Defense Science and Engineering Graduate (NDSEG) fellowship, and K.P.Y. in part by a National Science Foundation (NSF) Graduate Fellowship.Attached Files
Accepted Version - nihms633816.pdf
Supplemental Material - cbic_201402184_sm_miscellaneous_information.pdf
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Additional details
- PMCID
- PMC4199383
- Eprint ID
- 47532
- Resolver ID
- CaltechAUTHORS:20140728-141903775
- R01 GM62523
- NIH
- National Defense Science and Engineering Graduate (NDSEG) Fellowship
- NSF Graduate Research Fellowship
- Created
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2014-07-28Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field