title: Higher efficiency soluble prokaryotic expression, purification, and structural analysis of antimicrobial peptide G13.
abstract: 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-β-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 °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 β-sheet and random coil. Agar plate diffusion assay revealed that the r-G13 exhibited antibacterial activity against both Bacillus subtilis and E. coli.
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui, 230601, PR China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Che
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui, 230601, PR China.
authors.forename: Yinghu
authors.initials: Y
authors.lastname: Lu
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui, 230601, PR China. Electronic address: xdcha@163.com.
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Zha
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, PR China.
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, PR China. Electronic address: yangpeilong@caas.cn.
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui, 230601, PR China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Ma
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui, 230601, PR China.
authors.forename: Xuejiao
authors.initials: X
authors.lastname: Xu
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects; D001483:Base Sequence; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000187:drug effects; D015870:Gene Expression; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility
chemical_list: D000900:Anti-Bacterial Agents; D000945:Antigens, Differentiation, T-Lymphocyte; D023181:Antimicrobial Cationic Peptides; C075005:GNLY protein, human; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins
keywords: Antimicrobial peptide; Asp–Pro cleavage; Circular dichroism analysis; G13; Purification