Combinatorial codon-based amino acid substitutions
AUTOR(ES)
Yáñez, Jorge
FONTE
Oxford University Press
RESUMO
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 β-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 β-lactamase omega loop region.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=534637Documentos Relacionados
- Codon-based mutagenesis using dimer-phosphoramidites.
- Protein evolution by codon-based random deletions
- Novel ceftazidime-resistance β-lactamases generated by a codon-based mutagenesis method and selection
- Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols
- Predicting Deleterious Amino Acid Substitutions