Protein evolution by codon-based random deletions -: art. no. e136

被引：15

作者：

Osuna, J ^{[1
]}

Yáñez, J ^{[1
]}

Soberón, X ^{[1
]}

Gaytán, P ^{[1
]}

机构：

[1] Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Ingn Celular & Biocatalisis, Cuernavaca 62250, Morelos, Mexico

来源：

NUCLEIC ACIDS RESEARCH | 2004年 / 32卷 / 17期

关键词：

D O I：

10.1093/nar/gnh135

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

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