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High-yield soluble expression and simple purification of the antimicrobial peptide OG2 using the intein system in Escherichia coli.

Xie YG, Han FF, Luan C, Zhang HW, Feng J, Choi YJ, Groleau D, Wang YZ - Biomed Res Int (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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.

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Optimization of protein expression. lane M: SeeBlue Plus2 Pre-Stained Standard (kDa); lane UN: uninduced culture; lane IN: induced culture; lane SP: soluble protein; lane IB: inclusion body.
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fig2: Optimization of protein expression. lane M: SeeBlue Plus2 Pre-Stained Standard (kDa); lane UN: uninduced culture; lane IN: induced culture; lane SP: soluble protein; lane IB: inclusion body.

Mentions: Protein bands of approximately 30.3 kDa and 27.5 kDa, corresponding to the molecular weights of OG2-intein2 and intein1-OG2, respectively, were observed on the SDS-PAGE gel after induction (Figure 2, shown by arrows). Only 33% of the expressed OG2-intein2 was soluble after induction with 0.5 mM IPTG at 37°C. When the IPTG concentration was decreased to 0.1 mM and the induction temperature was lowered to 30°C, nearly 70% of the expressed OG2-intein2 became soluble. Although a low temperature (20°C) and a low IPTG concentration (0.1 mM) also enhanced the solubility of intein1-OG2, approximately 58% of the protein remained as insoluble. Therefore, induction with 0.1 mM IPTG for 2 h at 30°C and induction with 0.1 mM IPTG for 6 h at 20°C were used in the following experiments as optimum conditions for expression of OG2-intein2 and intein1-OG2, respectively.


High-yield soluble expression and simple purification of the antimicrobial peptide OG2 using the intein system in Escherichia coli.

Xie YG, Han FF, Luan C, Zhang HW, Feng J, Choi YJ, Groleau D, Wang YZ - Biomed Res Int (2013)

Optimization of protein expression. lane M: SeeBlue Plus2 Pre-Stained Standard (kDa); lane UN: uninduced culture; lane IN: induced culture; lane SP: soluble protein; lane IB: inclusion body.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3713655&req=5

fig2: Optimization of protein expression. lane M: SeeBlue Plus2 Pre-Stained Standard (kDa); lane UN: uninduced culture; lane IN: induced culture; lane SP: soluble protein; lane IB: inclusion body.
Mentions: Protein bands of approximately 30.3 kDa and 27.5 kDa, corresponding to the molecular weights of OG2-intein2 and intein1-OG2, respectively, were observed on the SDS-PAGE gel after induction (Figure 2, shown by arrows). Only 33% of the expressed OG2-intein2 was soluble after induction with 0.5 mM IPTG at 37°C. When the IPTG concentration was decreased to 0.1 mM and the induction temperature was lowered to 30°C, nearly 70% of the expressed OG2-intein2 became soluble. Although a low temperature (20°C) and a low IPTG concentration (0.1 mM) also enhanced the solubility of intein1-OG2, approximately 58% of the protein remained as insoluble. Therefore, induction with 0.1 mM IPTG for 2 h at 30°C and induction with 0.1 mM IPTG for 6 h at 20°C were used in the following experiments as optimum conditions for expression of OG2-intein2 and intein1-OG2, respectively.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus