Incorporation of Aliphatic Proline Residues into Recombinantly Produced Insulin
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1.
California Institute of Technology
Abstract
Analogs of proline can be used to expand the chemical space about the residue while maintaining its uniquely restricted conformational space. Here, we demonstrate the incorporation of 4R-methylproline, 4S-methylproline, and 4-methyleneproline into recombinant insulin expressed in Escherichia coli. These modified proline residues, introduced at position B28, change the biophysical properties of insulin: Incorporation of 4-methyleneproline at B28 accelerates fibril formation, while 4-methylation speeds dissociation from the pharmaceutically formulated hexamer. This work expands the scope of proline analogs amenable to incorporation into recombinant proteins and demonstrates how noncanonical amino acid mutagenesis can be used to engineer the therapeutically relevant properties of protein drugs.
Copyright and License
© 2023 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0.
Acknowledgement
We sincerely thank C. Paavola, M. Akers, J. Moyers, and A. Mahdavi for advice on proinsulin refolding. A. Lopez assisted with cloning proS mutants, and A. Chapman performed pilot ncPro incorporation studies. Circular dichroism spectroscopy was performed at the Beckman Institute Laser Resource Center at Caltech with assistance from J. Winkler. Electron microscopy was performed at the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech with input from S. Chen. Mass spectrometry was performed at the Caltech Multiuser Mass Spectrometry Laboratory in the Division of Chemistry and Chemical Engineering with the help of M. Shahgholi and at the Proteome Exploration Laboratory at Caltech with assistance from B. Quan. This work was supported by grants from the National Institutes of Health (1R01GM134013-01 and 1R01GM120600), the Juvenile Diabetes Research Foundation (3-IND-2015-118-I-X), the Canada 150 Research Chairs program (C150-2017-00015), the Canada Foundation for Innovation (CFI-37589), and the Canadian Natural Science and Engineering Research Council (DG-RGPIN-2019-05637). UltraScan supercomputer calculations were supported through NSF/XSEDE grant TG-MCB070039N and University of Texas grant TG457201. S.L.B. was supported by a National Science Foundation Graduate Research Fellowship under grant number 1745301. The Canadian Natural Science and Engineering Research Council supports A.H. through a scholarship grant.
Conflict of Interest
The authors declare the following competing financial interest(s): D.T. is an inventor on U.S. patents that describe the use of non-canonical amino acids in the engineering of insulin and other proteins.
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Additional details
- ISSN
- 1554-8937
- DOI
- 10.1021/acschembio.3c00561
- PMCID
- PMC10728891
- 1R01GM134013-01
- National Institutes of Health
- 1R01GM120600
- National Institutes of Health
- 3-IND-2015-118-I-X
- Juvenile Diabetes Research Foundation
- C150-2017-00015
- Canada 150 Research Chairs
- CFI-37589
- Canada Foundation for Innovation
- DG-RGPIN-2019-05637
- Natural Sciences and Engineering Research Council
- TG-MCB070039N
- National Science Foundation
- TG457201
- National Science Foundation
- NSF Graduate Research Fellowship DGE-1745301
- National Science Foundation