Expanded chemical diversity sampling through whole protein evolution

Baldwin, Amy Joy ORCID: https://orcid.org/0000-0002-2162-3771, Arpino, James Alexander, Edwards, W. R., Tippmann, Eric Michael and Jones, Darran Dafydd ORCID: https://orcid.org/0000-0001-7709-3995 2009. Expanded chemical diversity sampling through whole protein evolution. Molecular Biosystems 5 (7) , pp. 764-766. 10.1039/B904031E

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Abstract

A directed evolution method has been developed that allows random substitution of a contiguous trinucleotide sequence for TAG throughout a target gene for use in conjunction with an expanded genetic code. Using TEM-1 β-lactamase and enhanced green fluorescent protein as targets, protein variants were identified whose functional phenotype was rescued in vivo when co-expressed with orthogonal tRNA–aminoacyl-tRNA synthase pairs that insert p-iodophenylalanine in response to UAG. Sequencing of the selected clones that retained the target protein function revealed that >90% of the variants contained in-frame TAG codons distributed throughout the target gene. Such an approach will allow broader sampling of new chemical diversity by proteins, so opening new avenues for studying biological systems and for adapting proteins for biotechnological applications. A common set of reagents allows the method to be used on different protein systems and in combination with an array of different unnatural amino acids, so helping to reveal the true potential for engineering proteins through expanded chemical diversity sampling.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences Chemistry
Additional Information:	Pdf uploaded in accordance with publisher's policy at http://www.sherpa.ac.uk/romeo/issn/1742-206X/ (accessed 18.09.14).
Publisher:	Royal Society of Chemistry
ISSN:	1742-206X
Date of First Compliant Deposit:	30 March 2016
Last Modified:	12 May 2023 17:04
URI:	https://orca.cardiff.ac.uk/id/eprint/8799

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