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

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 SubSeq(NNN). The bla gene encoding TEM-1 beta-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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Baldwin AJ,Busse K,Simm AM,Jones DD

doi

10.1093/nar/gkn358

subject

Has Abstract

pub_date

2008-08-01 00:00:00

pages

e77

issue

13

eissn

0305-1048

issn

1362-4962

pii

gkn358

journal_volume

36

pub_type

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