Effects of thioredoxin: Sumo and intein on soluble fusion expression of an antimicrobial peptide OG2 in escherichia coli

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TypeArticle
Journal titleProtein and Peptide Letters
ISSN0929-8665
Volume20
Issue1
Pages5460; # of pages: 7
Subjectintein; polypeptide antibiotic agent; polypeptide antibiotic agent OG2; SUMO protein; thioredoxin; unclassified drug; affinity chromatography; article; chemical structure; controlled study; enzyme release; Escherichia coli; expression vector; hemolysis; molecular cloning; nonhuman; polymerase chain reaction; process optimization; protein expression; protein purification; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Erythrocytes; Escherichia coli; Hemolytic Agents; Inteins; Ranidae; Recombinant Fusion Proteins; Small Ubiquitin-Related Modifier Proteins; Staphylococcus aureus; Swine; Thioredoxins
AbstractOG2 is a modified antimicrobial peptide of Palustrin-OG1 (OG1), which is derived from Odorrana grahami frog. OG2 has shown much higher selective antimicrobial activity and lower hemolytic activity than OG1, indicating OG2 may be a promising antimicrobial agent. In this study, we investigated three fusion partners, including thioredoxin, Mxe GyrA intein, and small ubiquitin-like modifier (SUMO), each fused with OG2, and examined their effects on the expression level and solubility of OG2 in Escherichia coli. The codon-optimized OG2 gene was cloned into pET32a (+) and pTWIN1 for fusion with thioredoxin and Mxe GyrA intein, respectively. In addition, the SUMO-OG2 gene was amplified by splice overlap extension PCR method and was cloned into pET30a (+). All recombinant plasmids were then transformed into E. coli BL21(DE3)pLysS, and the expressed fusion proteins were verified. Upon isopropyl α-D-1- thiogalactopyranoside (IPTG) induction, OG2 fused with thioredoxin (Trx-OG2) showed the highest yield as a soluble fusion protein (50 mg/L), followed by Mxe GyrA intein (44 mg/L) and SUMO (11 mg/L). The thioredoxin-fused protein (Trx-OG2) was then purified by nickel-nitrilotriacetic acid chromatography and desalted by Sephadex G25. The OG2 released by both tobacco etch virus protease and enterokinase from Trx-OG2 showed strong antimicrobial activity against Staphylococcus aureus ATCC25923. Copyright © 2012 Bentham Science Publishers. All Rights Reserved.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Biotechnology Research Institute (BRI-IRB)
Peer reviewedYes
NPARC number21269899
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Record identifier71d61e3e-3517-4438-a81a-475c6f4fb1da
Record created2013-12-13
Record modified2016-05-09
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