Expression, purification and detection of activities of AprA and its inhibitor AprI from Pseudomonas aeruginosa
10.7644/j.issn.1674-9960.2016.12.008
- VernacularTitle:铜绿假单胞菌碱性蛋白酶AprA及其抑制剂AprI的表达、纯化和初步的活性验证
- Author:
Run CAO
;
Yaping GAO
;
Yu LIU
;
Chenghua LIU
;
Peng JIN
;
Yuanfang MA
;
Guang YANG
- Keywords:
Pseudomonas aeruginosa;
alkaline protease(AprA);
inhibitor of alkaline protease(AprI);
flagellin
- From:
Military Medical Sciences
2016;40(12):974-977,1012
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone and express the alkaline protease AprA , one important virulence factor secreted by Pseudomonas aeruginosa(PAE)in Escherichia coli, to clone and express the inhibitor of AprA (AprI) and its substrate flagellin , and to detect the function of AprA and the inhibitory function of AprI .Methods The genes encoding AprA ,AprI and flagellin gene were amplified respectively by PCR using PAE PAO 1 genome DNA as the template .The expression vec-tors (pET-28a-AprA, pET-28a-AprI and pET-28a-Flagellin) were constructed and transformed into E.coli BL21(DE3) respectively.The recombinant AprA protein was expressed by IPTG induction and purified via denaturing and renaturation. The recombinant AprI and flagellin were expressed and purified by Ni 2+affinity chromatography .The cleavage activities of AprA on flagellin were detected by SDS-PAGE.Results Recombinant AprA , AprI and flagellin protein were expressed and purified .It was demonstrated that AprA cleaved flagellin , which was blocked by AprI .Conclusion Recombinant AprA could cleave its substrates as an alkaline protease , and its inhibitor AprI inhibits the activities of AprA .This study will contribute to further investigations on the role of AprA in the pathogenesis of PAE .
