Expression and characterization of recombinant flagellin FlgE of Pseudomonas aeruginosa / 中华微生物学和免疫学杂志

ABSTRACT

Objective To construct the prokaryotic expression and purification protocols for Pseudomonas aeruginosa type E flagellin (FlgE) and to study its bioactivity.Methods With analysis of Pseudomonas aeruginosa flagellin FlgE sequences,the whole-length FlgE gene was amplified from Pseudomonas aeruginosa genomic DNA by using PCR and primers with proper restriction enzyme sites.The amplified FlgE fragment and prokaryotic expression plasmid pET24a were digested with Nde Ⅰ and Hind ⅢⅣ respectively.The target fragment and vector were recovered and ligated to obtain the recombinant plasmid pET24a-FlgE.DNA sequencing of positive clone confirmed that the target gene and the junctions with vectors were all correct.The plasmid pET24a-FlgE was transformed into BL21 bacteria.The culture conditions like temperature,rotation speed,inducer concentrations,time length were optimized to achieve maximal expression of the target recombinant FlgE with 6×His tag at C terminal.FlgE-His proteins were purified using His-Trap affinity chromatography columns and identified by SDS-PAGE.The purified proteins were further subjected to endotoxin elimination with proper kits.The purified recombinant FlgE was added to cultured corneal epithelial cells for 4 h and the expression of several inflammation-related molecules was examined by using real-time quantitative PCR.Results The recombinant plasmid pET24a-FlgE was successfully constructed and high level FlgE expression was achieved in BL21 with rotation at 16℃ and 1 mmol/L isopropyl-β-D-glucopyranosyl-galactosidase induction for 20 h.Purified recombinant FlgE-His was obtained and used for primary bioactivity assay.After treatment of corneal epithelial cells with 20 μg/ml FlgE for 4 h,the expression of inflammatory cytokines IL-6,IL-8 were significantly increased.Inactivation of the FlgE with ethanol abolished its stimulatory activity.Conclusion The prokaryotic expression and purification system for recombinant Pseudomonas aeruginosa flagellin FlgE was set up,and the recombinant FlgE stimulated the expression of inflammatory factors in corneal epithelial cells.