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

作者：Che, Yuanyuan; Lu, Yinghu; Zha, Xiangdong^*; Huang, Huoqing; Yang, Peilong^*; Ma, Lijuan; Xu, Xuejiao

来源：Protein Expression and Purification, 2016, 119: 45-50.

DOI：10.1016/j.pep.2015.11.006

• G13
• Antimicrobial peptide
• Asp-Pro cleavage
• Purification
• Circular dichroism analysis

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.

• 出版日期2016-3
• 单位安徽大学; 中国农业科学院