Recombinant expression, refolding, purification and characterization of Pseudomonas aeruginosa protease IV in Escherichia coli

Protein Expr Purif. 2016 Oct:126:69-76. doi: 10.1016/j.pep.2016.05.019. Epub 2016 May 31.

Abstract

Several protease IV enzymes are widely used in proteomic research. Specifically, protease IV from Pseudomonas aeruginosa has lysyl endopeptidase activity. Here, we report the recombinant expression, refolding, activation, and purification of this protease in Escherichia coli. Proteolytic instability of the activated intermediate, a major obstacle for efficient production, is controlled through ammonium sulfate precipitation. The purified protease IV exhibits superior lysyl endopeptidase activity compared to a commercial product.

Keywords: Activity; Protease IV; Pseudomonas aeruginosa; Purification; Recombinant expression; Refolding.

MeSH terms

  • Bacterial Proteins* / biosynthesis
  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / isolation & purification
  • Gene Expression*
  • Peptide Hydrolases* / biosynthesis
  • Peptide Hydrolases* / chemistry
  • Peptide Hydrolases* / genetics
  • Peptide Hydrolases* / isolation & purification
  • Protein Refolding*
  • Pseudomonas aeruginosa* / enzymology
  • Pseudomonas aeruginosa* / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • Peptide Hydrolases
  • protease IV