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Nucleic Acids Research Advance Access originally published online on June 17, 2008
Nucleic Acids Research 2008 36(13):e77; doi:10.1093/nar/gkn358
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Nucleic Acids Research, 2008, Vol. 36, No. 13 e77
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Amy J. Baldwin, Kathy Busse, Alan M. Simm and D. Dafydd Jones*

School of Biosciences, Cardiff University, UK

*To whom correspondence should be addressed. Tel: +44 0 29 2087 4290; Fax: +44 0 29 2087 4116; Email: jonesdd{at}cf.ac.uk

Received March 19, 2008. Revised May 14, 2008. Accepted May 20, 2008.

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.

Present addresses: Kathy Busse, Institut für Biochemie der Med. Fakultät, Universität Leipzig, Leipzig, Germany

Alan M. Simm, School of Engineering and Electronics, University of Edinburgh, Edinburgh, UK


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