Combinatorial codon-based amino acid substitutions.
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 beta-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 beta-lactamase omega loop region.
Yáñez
Jorge
J
Instituto de Biotecnología/UNAM, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.
Argüello
Martha
M
Osuna
Joel
J
Soberón
Xavier
X
Gaytán
Paul
P
D019943:Amino Acid Substitution; D002442:Ceftazidime / Q000494:pharmacology; D003062:Codon / Q000138:chemical synthesis* / Q000737:chemistry; D020650:Combinatorial Chemistry Techniques; D015723:Gene Library; D016296:Mutagenesis; D009943:Organophosphorus Compounds / Q000737:chemistry; D001618:beta-Lactamases / Q000235:genetics
D003062:Codon; D009943:Organophosphorus Compounds; C434331:phosphoramidite; D002442:Ceftazidime; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1