Secretory expression of active clostripain in Escherichia coli.
In this study, the clostripain gene was modified and its signal sequence was replaced with that of penicillin G acylase (PGA). The core clostripain protein fused to the PGA signal peptide was also prepared. With regard to the expression of the clostripain precursors, the majority of clostripain activity was observed in the culture media, thereby indicating that both the clostripain signal peptide and the PGA signal peptide were recognized in the E. coli secretion pathway, and the precursors successfully matured into the active form. Otherwise, the activity was rather low when the core protein was expressed, which indicates that the clostripain pro-peptide is important in the formation of the active enzyme in E. coli. Enzyme activity reached a value of 3200U/L in CGY media for high expression. The recombinant clostripain and porcine carboxypeptidase B were used in the conversion of a proinsulin fusion protein into insulin. The leader peptide (LP) and the proinsulin C-peptide appeared to have been removed simultaneously, and the final cleavage product evidenced an HPLC retention time identical to that of the insulin standard, thereby implying that the clostripain specifically cleaved the arginine residues in the LP and in the C-peptide. We have also demonstrated the possibility that the recombinant clostripain might prove useful in the production of insulin from the proinsulin fusion protein.
Kim
Chang-Kyu
CK
College of Animal Bioscience & Technology, Konkuk University, Seoul, Republic of Korea.
Lee
Sang-Yong
SY
Kwon
Oh-Joong
OJ
Lee
Sang-Mok
SM
Nah
Seung-Yeol
SY
Jeong
Sang Min
SM
D003001:Cloning, Molecular / Q000379:methods; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000138:chemical synthesis*; D011384:Proinsulin / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D007328:Insulin; D011993:Recombinant Fusion Proteins; D011384:Proinsulin; D003546:Cysteine Endopeptidases; C019913:clostripain