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PEDS Advance Access published online on June 17, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn034
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

A one-pot, simple methodology for cassette randomisation and recombination for focused directed evolution

Aurelio Hidalgo1,2, Anna Schließmann1, Rafael Molina3, Juan Hermoso3 and Uwe T. Bornscheuer1,4

1Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Ernst-Moritz-Arndt University Greifswald, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany 2Centro de Biología Molecular ‘Severo Ochoa’ (UAM-CSIC), Nicolas Cabrera 1, 28049 Madrid, 3Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química Física ‘Rocasolano’, Consejo Superior de Investigaciones Científicas (CSIC), Serrano 119, 28006 Madrid, Spain

4 To whom correspondence should be addressed. E-mail: uwe.bornscheuer{at}uni-greifswald.de; web: http://www.chemie.uni-greifswald.de/~biotech

Protein engineering is currently performed either by rational design, focusing in most cases on only a few positions modified by site-directed mutagenesis, or by directed molecular evolution, in which the entire protein-encoding gene is subjected to random mutagenesis followed by screening or selection of desired phenotypes. A novel alternative is focused directed evolution, in which only fragments of a protein are randomised while the overall scaffold of a protein remains unchanged. For this purpose, we developed a PCR technique using long, spiked oligonucleotides, which allow randomising of one or several cassettes in any given position of a gene. This method allows over 95% incorporation of mutations independently of their position within the gene, yielding sufficient product to generate large libraries, and the possibility of simultaneously randomising more than one locus at a time, thus originating recombination. The high efficiency of this method was verified by creating focused mutant libraries of Pseudomonas fluorescens esterase I (PFEI), screening for altered substrate selectivity and validating against libraries created by error-prone PCR. This led to the identification of two mutants within the OSCARR library with a 10-fold higher catalytic efficiency towards p-nitrophenyl dodecanoate. These PFEI variants were also modelled in order to explain the observed effects.

Keywords: esterase/focused directed evolution/megaprimer PCR/substrate selectivity

Received December 28, 2007; revised April 28, 2008; accepted May 19, 2008.


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