A one-pot, simple methodology for cassette randomisation and recombination for focused directed evolution.
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.
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Ernst-Moritz-Arndt University Greifswald, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany.
Aurelio
A
Hidalgo
Anna
A
Schliessmann
Rafael
R
Molina
Juan
J
Hermoso
Uwe T
UT
Bornscheuer
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D015723:Gene Library; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009841:Oligonucleotides; D043182:Carboxylesterase; C469486:Ces1c protein, mouse