Protein evolution by codon-based random deletions.
A method to delete in-phase codons throughout a defined target region of a gene has been developed. This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode. Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is based on the assembly of oligonucleotides and on the use of two transient orthogonal protecting groups during the chemical synthesis. The performance of the method for protein function evolution was demonstrated by changing the substrate specificity of TEM-1 beta-lactamase. Functional ceftazidime-resistant beta-lactamase variants containing several deleted residues inside the catalytically important omega-loop region were found. The results show that the COBARDE method is a useful new molecular tool to access previously unexplorable sequence space.
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología/UNAM, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.
Joel
J
Osuna
Jorge
J
Yáñez
Xavier
X
Soberón
Paul
P
Gaytán
D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009841:Oligonucleotides / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000235:genetics; D017384:Sequence Deletion; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D003062:Codon; D009841:Oligonucleotides; D011506:Proteins; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1