Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 754319, 6 pages http://dx.doi.org/10.1155/2013/754319 Research Article High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli Yong-Gang Xie, 1 Fei-Fei Han, 1 Chao Luan, 1 Hai-Wen Zhang, 1 Jie Feng, 1 Young-Jun Choi, 2 Denis Groleau, 3 and Yi-Zhen Wang 1 1 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 2 Biotechnology Research Institute, National Research Council, Montreal, QC, Canada H4P 2R2 3 Chemical and Biotechnological Engineering, University of Sherbrooke, Sherbrooke, QC, Canada J1K 2R1 Correspondence should be addressed to Yi-Zhen Wang; yzwang321@zju.edu.cn Received 6 February 2013; Accepted 14 June 2013 Academic Editor: Jong-Soo Lee Copyright © 2013 Yong-Gang Xie et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. OG2 is a modifed 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 difcult to achieve high expression yields and efcient purifcation. In this study, we achieved high-yield OG2 expression using the intein fusion system. Te 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 afer 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 100mM DTT. Intein1-OG2 was less amenable to cleavage due to the inhibition of cleavage by the N-terminal amino acid of OG2. Te purifed OG2 exhibited strong antimicrobial activity against E. coli K88. Te intein system is the best currently available system for the cost-efective production of OG2. 1. Introduction In general, antimicrobial peptides (AMPs) are small peptides (10–50 amino acids) with a net positive charge (generally +2 to +9) and a substantial proportion (≥30%) of hydrophobic residues [1]. Tey are distributed in a wide range of organisms from single-celled microorganisms to humans [2] and play important roles in host immune defense by direct inhibiting of bacteria, fungi, viruses, and parasites growth and by im- mune modulation. By doing so, AMPs are regarded as a new generation of antibiotics as well as innate immune modula- tors [1]. Amphibian skin, such as skin of the Odorrana grahami where 107 novel AMPs were discovered [3], is one of the most generous sources of AMPs. Te mature Palustrin-OG1 (OG1) is one of those peptides that showed high activ- ity against Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 at the concentration of 16 g/mL [3, 4]. However, the concentration of OG1 that induces 50% hemolysis of human erythrocytes (HC 50 ) was 49.6 g/mL [4], which limits the application of OG1 as a therapeutic agent. In such case, OG2 (KKFFLKVLTKIRCKVAGGCRT) was generated through amino acid deletions and substitutions from the sequence of OG1, and this newly designed OG2 showed higher net positive charge, higher amphiphilicity, and lower hydrophobicity than OG1. Since OG2 showed much lower cytotoxicity and higher antimicrobial activity than the parental peptide OG1, it could be applicable as a therapeutic agent [5].