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

TY - JOUR

T1 - A one-pot, simple methodology for cassette randomisation and recombination for focused directed evolution

AU - Hidalgo, Aurelio

AU - Schliessmann, Anna

AU - Molina, Rafael

AU - Hermoso, Juan

AU - Bornscheuer, Uwe T

PY - 2008/9

Y1 - 2008/9

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Bacterial Proteins/chemistry

KW - Carboxylesterase/chemistry

KW - Directed Molecular Evolution

KW - Gene Library

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Oligonucleotides/metabolism

KW - Polymerase Chain Reaction

KW - Pseudomonas fluorescens/enzymology

KW - Substrate Specificity

U2 - 10.1093/protein/gzn034

DO - 10.1093/protein/gzn034

M3 - Journal article

C2 - 18559369

SN - 1741-0126

VL - 21

SP - 567

EP - 576

JO - Protein Engineering, Design and Selection

JF - Protein Engineering, Design and Selection

IS - 9

ER -