Higher efficiency soluble prokaryotic expression, purification, and structural analysis of antimicrobial peptide G13

被引：6

作者：

Che, Yuanyuan ^{[1
]}

Lu, Yinghu ^{[1
]}

Zha, Xiangdong ^{[1
]}

Huang, Huoqing ^{[2
]}

Yang, Peilong ^{[2
]}

Ma, Lijuan ^{[1
]}

Xu, Xuejiao ^{[1
]}

机构：

[1] Anhui Univ, Sch Life Sci, Hefei 230601, Anhui, Peoples R China

[2] Chinese Acad Agr Sci, Feed Res Inst, Key Lab Feed Biotechnol, Minist Agr, Beijing 100081, Peoples R China

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2016年 / 119卷

关键词：

G13; Antimicrobial peptide; Asp-Pro cleavage; Purification; Circular dichroism analysis; HOST-DEFENSE PEPTIDES; FUSION;

D O I：

10.1016/j.pep.2015.11.006

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

G13 is a 19-residue cationic antimicrobial peptide derived from granulysin. In order to achieve high-level expression of G13 in Escherichia coli cells, and to reduce downstream processing costs, we introduced an Asp Pro acid labile bond between the His-Patch thioredoxin and G13 and constructed the recombinant plasmid pThiohisA-DP-G13. The plasmid was transformed into E. coli BL21 (DE3). After induction with isopropyl-beta-D-thiogalactopyranoside for 5 h, the fusion protein accumulated up to 200 mg/L in soluble form. The fusion protein was released by a high pressure homogenizer, cleaved using 13% acetic acid at 50 degrees C hydrolysis for 72 h. The recombinant G13 (r-G13) was then successively purified by fractional precipitation with ammonium sulfate and trichloroacetic acid, followed by one-step cation exchange chromatography. The purified r-G13 displayed a single band (about 2.2 kDa) as analyzed by Tris Tricine buffered SDS-PAGE, and its precise molecular weight was confirmed using tandem mass spectrometry. Analysis of r-G13 by circular dichroism (CD) indicated that r-G13 contained predominantly beta-sheet and random coil. Agar plate diffusion assay revealed that the r-G13 exhibited antibacterial activity against both Bacillus subtilis and E. coli. (C) 2015 Elsevier Inc. All rights reserved.

引用

页码：45 / 50

页数：6

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