Functional expression of a plant hydroxynitrile lyase in Escherichia coli by directed evolution: creation and characterization of highly in vivo soluble mutants

Yasuhisa Asano, Mohammad Dadashipour, Mizue Yamazaki, Nobutaka Doi, Hidenobu Komeda

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

Abstract

Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering. Structure-guided saturation mutagenesis caused high solubility of single Lys-Pro mutations at positions 176, 199 and 224 of this low soluble wild-type enzyme. The triple Lys-Pro mutant generated at these surface conserved residues showed up to 8-fold increase in specific activity in the cell-free extract. Random mutagenesis also created a mutant of His103Met with 18.5-fold increase. The main expression form was reversed from insoluble to the soluble fraction following both types of above-mentioned mutations in E.coli at 37°C. The findings challenge the rationale of producing recombinant proteins in this host at 37°C. Formerly wild type low soluble protein was then present as soluble protein by these mutations, which also elevated the total soluble protein fraction in E.coli. Saturation mutagenesis of His103 provided other highly soluble mutants with hydrophobic substitutions. These mutations caused only minor secondary structural changes as determined by circular dichroism and Fourier-transform infrared spectroscopy and affected catalytic efficiency slightly for the purified mutants (0.82-1.6-fold for benzaldehyde and 0.9-1.9-fold for mandelonitrile). The stability of the mutants was differed from that of the wild type at high temperatures and at pH >8. Exchanging the buried basic-polar residue His103 with hydrophobic amino acids is in line with the overall structure of the enzyme, i.e. having hydrophilic residues in solvent-exposed areas and hydrophobic residues in the core.

Original languageEnglish
Pages (from-to)607-16
Number of pages10
JournalProtein engineering, design & selection : PEDS
Volume24
Issue number8
DOIs
Publication statusPublished - Aug 2011

Keywords

  • Aldehyde-Lyases/biosynthesis
  • Amino Acid Sequence
  • Circular Dichroism
  • Directed Molecular Evolution/methods
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli/genetics
  • Hydrogen-Ion Concentration
  • Manihot/enzymology
  • Molecular Sequence Data
  • Mutation
  • Plant Proteins/biosynthesis
  • Protein Engineering/methods
  • Protein Stability
  • Recombinant Proteins/biosynthesis
  • Sequence Alignment
  • Solubility
  • Spectrometry, Fluorescence
  • Spectroscopy, Fourier Transform Infrared
  • Temperature

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