Recombinant expression, refolding, purification and characterization of Pseudomonas aeruginosa protease IV in Escherichia coli.
Several protease IV enzymes are widely used in proteomic research. Specifically, protease IV from Pseudomonas aeruginosa has lysyl endopeptidase activity. Here, we report the recombinant expression, refolding, activation, and purification of this protease in Escherichia coli. Proteolytic instability of the activated intermediate, a major obstacle for efficient production, is controlled through ammonium sulfate precipitation. The purified protease IV exhibits superior lysyl endopeptidase activity compared to a commercial product.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, PR China.
Mingzhi
M
Zhao
Prosit Sole Biotechnology, Co., Ltd., Beijing, 100085, PR China.
Man
M
Cai
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, PR China; Life Science College, Southwest Forestry University, Kunming, 650224, PR China.
Feilin
F
Wu
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, PR China; Institute of Microbiology Chinese Academy of Science, Beijing, 100101, PR China.
Yao
Y
Zhang
Life Science College, Southwest Forestry University, Kunming, 650224, PR China.
Zhi
Z
Xiong
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, PR China; Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, PR China; Anhui Medical University, Hefei, 230032, Anhui, PR China. Electronic address: xuping_bprc@126.com.
Ping
P
Xu
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D058849:Protein Refolding; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; C030732:protease IV
Activity; Protease IV; Pseudomonas aeruginosa; Purification; Recombinant expression; Refolding