High-yield soluble expression and simple purification of the antimicrobial peptide OG2 using the intein system in Escherichia coli.
OG2 is a modified antimicrobial peptide, that is, derived from the frog peptide Palustrin-OG1. It has high antimicrobial activity and low cytotoxicity, and it is therefore promising as a therapeutic agent. Both prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) production host systems were used to produce OG2 in our previous study; however, it was difficult to achieve high expression yields and efficient purification. In this study, we achieved high-yield OG2 expression using the intein fusion system. The optimized OG2 gene was cloned into the pTWIN1 vector to generate pTWIN-OG2-intein2 (C-terminal fusion vector) and pTWIN-intein1-OG2 (N-terminal fusion vector). Nearly 70% of the expressed OG2-intein2 was soluble after the IPTG concentration and induction temperature were decreased, whereas only 42% of the expressed of intein1-OG2 was soluble. Up to 75 mg of OG2-intein2 was obtained from a 1l culture, and 85% of the protein was cleaved by 100 mM DTT. Intein1-OG2 was less amenable to cleavage due to the inhibition of cleavage by the N-terminal amino acid of OG2. The purified OG2 exhibited strong antimicrobial activity against E. coli K88. The intein system is the best currently available system for the cost-effective production of OG2.
Key Laboratory of Animal Nutrition and Feed Science of Ministry of Agriculture, Key Laboratory of Feed and Animal Nutrition of Zhejiang Province, Institute of Feed Science, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Yong-Gang
YG
Xie
Fei-Fei
FF
Han
Chao
C
Luan
Hai-Wen
HW
Zhang
Jie
J
Feng
Young-Jun
YJ
Choi
Denis
D
Groleau
Yi-Zhen
YZ
Wang
D000818:Animals; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D047668:Inteins / Q000235:genetics*; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; C581684:palustrin-OG1, Odorrana grahami