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Nucleic Acids Research 2004 32(17):e136; doi:10.1093/nar/gnh135
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Published online 30 September 2004

Nucleic Acids Research, Vol. 32 No. 17 © Oxford University Press 2004; all rights reserved

Protein evolution by codon-based random deletions

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

Departamento de Ingeniería Celular y Biocatálisis, 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 August 13, 2004; Revised September 10, 2004; Accepted September 15, 2004

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 ß-lactamase. Functional ceftazidime-resistant ß-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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