High-level expression of pseudolysin, the extracellular elastase of Pseudomonas aeruginosa, in Escherichia coli and its purification

• Published in: Protein expression and purification 2015-09, Vol.113, p.79-84
• Main Authors: Odunuga, Odutayo O., Adekoya, Olayiwola A., Sylte, Ingebrigt
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Cloning, Molecular; Elastase; Escherichia coli - genetics; Inclusion Bodies; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Metalloendopeptidases - isolation & purification; Metalloendopeptidases - metabolism; Over-expression; Pancreatic Elastase; Precipitation; Protein Refolding; Pseudolysin; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Purification; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Refolding
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.05.005

•Full-length pseudolysin was successfully expressed at high levels in Escherichia coli.•Full-length pseudolysin was purified in relatively large quantity.•Denatured pseudolysin was refolded correctly without zinc and calcium and shown to be catalytically active.•Precipitation during refolding of urea-denatured pseudolysin was eliminated.•A fast and reproducible scheme for expression and purification of pseudolysin was developed. Pseudolysin is the extracellular elastase of Pseudomonas aeruginosa and belongs to the thermolysin-like family of metallopeptidases. Pseudolysin has been identified as a robust drug target and a biotechnologically important enzyme in the tanning industry. Previous attempts to purify active pseudolysin from P. aeruginosa or by expression in Escherichia coli yielded low quantities. Considerable expression and purification of secreted pseudolysin from Pichia pastoris has been reported but it is time-consuming and not cost-effective. We report the successful large-scale expression of pseudolysin in E. coli and purification of the correctly folded and active protein. The lasB gene that codes for the enzymatically active mature 33-kilodalton pseudolysin was expressed with a histidine tag under the control of the T7 promoter. Pseudolysin expressed highly in E. coli and was solubilized and purified in 8M urea by metal affinity chromatography. The protein was simultaneously further purified, refolded and buffer-exchanged on a preparative Superdex 200 column by a modified urea reverse-gradient size exclusion chromatography. Using this technique, precipitation of pseudolysin was completely eliminated. Refolded pseudolysin was found to be active as assessed by its ability to hydrolyze N-succinyl-ala-ala-ala-p-nitroanilide. The purification scheme yielded approximately 40mg of pseudolysin per liter of expression culture and specific activity of 3.2U/mg of protein using N-succinyl-ala-ala-ala-p-nitroanilide as substrate. This approach provides a reproducible strategy for high-level expression and purification of active metallopeptidases and perhaps other inclusion body-forming and precipitation-prone proteins.