title: High-level expression of pseudolysin, the extracellular elastase of Pseudomonas aeruginosa, in Escherichia coli and its purification.
abstract: 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 40 mg 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.
authors.affiliation: Department of Chemistry and Biochemistry, Stephen F. Austin State University, PO Box 13006, SFA Station, Nacogdoches, TX 75962, USA. Electronic address: odunugaoo@sfasu.edu.
authors.forename: Odutayo O
authors.initials: OO
authors.lastname: Odunuga
authors.affiliation: Department of Pharmacy, Faculty of Health Sciences, UiT The Arctic University of Norway, NO-9037 Tromsø, Norway.
authors.forename: Olayiwola A
authors.initials: OA
authors.lastname: Adekoya
authors.affiliation: Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, NO-9037 Tromsø, Norway.
authors.forename: Ingebrigt
authors.initials: I
authors.lastname: Sylte
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D002479:Inclusion Bodies; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010196:Pancreatic Elastase; D058849:Protein Refolding; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa
keywords: Elastase; Over-expression; Precipitation; Pseudolysin; Purification; Refolding