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

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

ISBN:

1741-0134

Call Number:

18559369

Accession Number:

18559369

URL:

http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=18559369&retmode=ref&cmd=prlinks

Keywords:

Amino Acid Sequence; Bacterial Proteins; Carboxylesterase; Directed Molecular Evolution; Gene Library; Models; Molecular; Molecular Sequence Data; Mutagenesis; Site-Directed; Oligonucleotides; Polymerase Chain Reaction; Pseudomonas fluorescens; Substrate Specificity

Abstract:

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.