In-frame amber stop codon replacement mutagenesis for the directed evolution of proteins containing non-canonical amino acids: identification of residues open to bio-orthogonal modification

Arpino, James A. J., Baldwin, Amy Joy ORCID: https://orcid.org/0000-0002-2162-3771, McGarrity, Adam R., Tippmann, Eric M. and Jones, Darran Dafydd ORCID: https://orcid.org/0000-0001-7709-3995 2015. In-frame amber stop codon replacement mutagenesis for the directed evolution of proteins containing non-canonical amino acids: identification of residues open to bio-orthogonal modification. PLoS ONE 10 (5) , e0127504. 10.1371/journal.pone.0127504

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Abstract

Expanded genetic code approaches are a powerful means to add new and useful chemistry to proteins at defined residues positions. One such use is the introduction of non-biological reactive chemical handles for site-specific biocompatible orthogonal conjugation of proteins. Due to our currently limited information on the impact of non-canonical amino acids (nAAs) on the protein structure-function relationship, rational protein engineering is a “hit and miss” approach to selecting suitable sites. Furthermore, dogma suggests surface exposed native residues should be the primary focus for introducing new conjugation chemistry. Here we describe a directed evolution approach to introduce and select for in-frame codon replacement to facilitate engineering proteins with nAAs. To demonstrate the approach, the commonly reprogrammed amber stop codon (TAG) was randomly introduced in-frame in two different proteins: the bionanotechnologically important cyt b562 and therapeutic protein KGF. The target protein is linked at the gene level to sfGFP via a TEV protease site. In absence of a nAA, an in-frame TAG will terminate translation resulting in a non-fluorescent cell phenotype. In the presence of a nAA, TAG will encode for nAA incorporation so instilling a green fluorescence phenotype on E. coli. The presence of endogenously expressed TEV proteases separates in vivo target protein from its fusion to sfGFP if expressed as a soluble fusion product. Using this approach, we incorporated an azide reactive handle and identified residue positions amenable to conjugation with a fluorescence dye via strain-promoted azide-alkyne cycloaddition (SPAAC). Interestingly, best positions for efficient conjugation via SPAAC were residues whose native side chain were buried through analysis of their determined 3D structures and thus may not have been chosen through rational protein engineering. Molecular modeling suggests these buried native residues could become partially exposed on substitution to the azide containing nAA.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Subjects:	Q Science > QR Microbiology
Additional Information:	This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Publisher:	Public Library of Science
ISSN:	1932-6203
Funders:	BBSRC
Date of First Compliant Deposit:	30 March 2016
Date of Acceptance:	30 April 2015
Last Modified:	15 Mar 2024 07:23
URI:	https://orca.cardiff.ac.uk/id/eprint/74082

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