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

Research output: Contribution to journalJournal articleResearchpeer-review

  • Aurelio Hidalgo
  • Anna Schliessmann
  • Molina, Rafael
  • Juan Hermoso
  • Uwe T Bornscheuer

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.

Original languageEnglish
JournalProtein Engineering Design and Selection (Print)
Volume21
Issue number9
Pages (from-to)567-76
Number of pages10
ISSN1741-0126
DOIs
Publication statusPublished - Sep 2008

    Research areas

  • Amino Acid Sequence, Bacterial Proteins/chemistry, Carboxylesterase/chemistry, Directed Molecular Evolution, Gene Library, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligonucleotides/metabolism, Polymerase Chain Reaction, Pseudomonas fluorescens/enzymology, Substrate Specificity

ID: 203019702