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Nucleic Acids Research 2004 32(20):e158; doi:10.1093/nar/gnh156
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Published online 10 November 2004

Nucleic Acids Research, Vol. 32 No. 20 © Oxford University Press 2004; all rights reserved

Combinatorial codon-based amino acid substitutions

Jorge Yáñez, Martha Argüello, Joel Osuna, Xavier Soberón and Paul Gaytán*

Instituto de Biotecnología/UNAM, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México

* To whom correspondence should be addressed. Tel: +52 777 329 1604; Fax: +52 777 317 2388; Email: paul{at}ibt.unam.mx

Received September 28, 2004; Revised and Accepted October 22, 2004

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 ß-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the ß-lactamase omega loop region.


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