Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx)

Baldwin, Amy Joy ORCID: https://orcid.org/0000-0002-2162-3771, Busse, K., Simm, A. M. and Jones, Darran Dafydd ORCID: https://orcid.org/0000-0001-7709-3995 2008. Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx). Nucleic Acids Research 36 (13) , e77. 10.1093/nar/gkn358

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Abstract

Trinucleotide exchange (TriNEx) is a method for generating novel molecular diversity during directed evolution by random substitution of one contiguous trinucleotide sequence for another. Single trinucleotide sequences were deleted at random positions in a target gene using the engineered transposon MuDel that were subsequently replaced with a randomized trinucleotide sequence donated by the DNA cassette termed SubSeqNNN. The bla gene encoding TEM-1 β-lactamase was used as a model to demonstrate the effectiveness of TriNEx. Sequence analysis revealed that the mutations were distributed throughout bla, with variants containing single, double and triple nucleotide changes. Many of the resulting amino acid substitutions had significant effects on the in vivo activity of TEM-1, including up to a 64-fold increased activity toward ceftazidime and up to an 8-fold increased resistance to the inhibitor clavulanate. Many of the observed amino acid substitutions were only accessible by exchanging at least two nucleotides per codon, including charge-switch (R164D) and aromatic substitution (W165Y) mutations. TriNEx can therefore generate a diverse range of protein variants with altered properties by combining the power of site-directed saturation mutagenesis with the capacity of whole-gene mutagenesis to randomly introduce mutations throughout a gene.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Subjects:	Q Science > QR Microbiology
Additional Information:	9 pp.
Publisher:	Oxford University Press
ISSN:	0305-1048
Funders:	BBSRC, Welsh Government
Date of First Compliant Deposit:	30 March 2016
Last Modified:	17 May 2023 06:21
URI:	https://orca.cardiff.ac.uk/id/eprint/8800

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