Cloning And Expression Of Protease IV Of Pseudomonas Aeruginosa

Pseudomonas aeruginosa proteaseⅣ(Ps-Ⅳ)is a lysine-specific endoprotease and causative agent secreted by most Pseudomonas aeruginosa strains isolated.It can specifically hydrolyze the carboxyl side of lysine-containing peptides and digest a number of biologically important proteins,such as fibrinogen,plasminogen,immunoglobulin G,and the complement proteins C1q and C3.Pseduomonas aeruginosa CMCC(B)10104 was studied in thesis.Using Chromozym PL as substrate,lysyl endopeptidase activity was found in the culture broths which proved that Pseduomonas aeruginosa CMCC(B)10104 maybe contain the Ps-Ⅳgene.According to the sequence of Ps-Ⅳin other Pseudomonas aeruginosa strains,appropriate primes were designed and synthesized.Ps-Ⅳgene was obtained from genomic DNA of Pseduomonas aeruginosa CMCC(B)10104.Subsequently,Ps-Ⅳgene was cloned into plasmid vector pET-28a and pNJUTRX, respectively.The recombined plasmid pET-28a-Tag(his)-Ps-Ⅳand pNJUTRX-Tag(his)-Ps-Ⅳwere transformed into E.coli BL21(DE3),and expressed by adding IPTG.Detected by SDS-PAGE analysis,Ps-Ⅳwas expressed in both expression systems.The Ps-Ⅳexpressed in precipitate(inclusion body)of pET-28a-Tag(his)-Ps-Ⅳ/ E.coli BL21 system and partially in supernatant of pNJUTRX-Tag(his)-Ps-Ⅳ/ E.coli BL21 system.The expression condition of pNJUTRX-Tag(his)-Ps-Ⅳ/E.coli BL21 was studied through orthogonal design,it indicated that incubation time after adding IPTG influenced the amount of soluble Ps-Ⅳ,while incubation temperature and the final concentration of IPTG have no significant effect.The soluble Ps-Ⅳexpressed in pNJUTRX-Tag(his)-Ps-Ⅳ/ E.coli BL21 system was purified by means of Metal chelate affinity chromatography、ion exchange chromatography and gel filtration chromatography.By Lowry method for protein quanification and lysyl endopeptidase activity assay,0.12 mg Ps-Ⅳcan be obtained from one liter medium,which contain 0.25U enzymatic activity.