Cloning, expression and purification of Pseudomonas aeruginosa pilin protein in the prokaryotic host

Background: Pathogenic Pseudomonas aeruginosa strains produce polar pili has required for motility, adhesion, and invasion. The main aims of the present study are to identify, clone, express and purify the recombinant pilin protein of P. aeruginosa in the prokaryotic host. Material and methods: The recombinant pilin gene (pilA) was isolated from P. aeruginosa PAO۱ strain by PCR and cloned into pET-۲۲b vector. The recombinant plasmid was subsequently verified by restriction analysis, and DNA sequencing. The recombinant vector was transformed into E. coli BL۲۱ (DE۳) strain, then the recombinant pilin overexpressed and affinity purified by Ni-NTA agarose-affinity chromatography.Western blot analysis was performed using anti-۶His tag antibody. Results: The PCR and enzymatic digestion results showed the accuracy of the pilA gene cloning. The protein electrophoresis showed that the molecular weight of recombinant pilin is about ۱۹ kDa. Western blot analysis also confirmed the production of recombinant protein. The amount of produced protein was measured by the direct spectrophotometery method, which was ۲/۵۸ mg/mL. Conclusion: Western blot and ELISA results along with that of sequencing ensure accurate production of recombinant pilin, retaining its partial epitopes.

Keywords:

Pseudomonas aeruginosa pilin protein , Cloning , Expression , پروتئین پیلین سودوموناس آئروژینوزا , کلونینگ , بیان