Effects of thioredoxin: SUMO and intein on soluble fusion expression of an antimicrobial peptide OG2 in Escherichia coli.
OG2 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 β-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.
Key Laboratory of Animal Nutrition and Feed Science of Ministry of Agriculture, Institute of Feed Science, Zhejiang University, Hangzhou, Zhejiang, PR China.
Yong-Gang
YG
Xie
Chao
C
Luan
Hai-Wen
HW
Zhang
Fei-Fei
FF
Han
Jie
J
Feng
Young-Jun
YJ
Choi
Denis
D
Groleau
Yi-Zhen
YZ
Wang
D000818:Animals; D000890:Anti-Infective Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis* / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects*; D053878:Hemolytic Agents / Q000494:pharmacology; D047668:Inteins; D011898:Ranidae; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000494:pharmacology*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000187:drug effects*; D013552:Swine; D013879:Thioredoxins / Q000737:chemistry* / Q000494:pharmacology
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D053878:Hemolytic Agents; C581685:OG2 peptide; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C581684:palustrin-OG1, Odorrana grahami; D013879:Thioredoxins