title: High-level expression of the recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) with an ubiquitin fusion partner in Escherichia coli.
abstract: The hybrid antibacterial peptide CA-MA [cecropinA(1-8)-magainin2(1-12)] with potent antimicrobial properties but no hemolytic activity is a potential alternative antibiotic. To explore a new approach for high-level expression of the hybrid peptide CA-MA in Escherichia coli, the sequence of ubiquitin (UBI) from housefly was inserted into the plasmid pQE30 to construct the vector pQEUBI. The cDNA fragment encoding CA-MA with preferred codons of E. coli was obtained by recursive PCR (rPCR) and cloned into the vector pQEUBI to express the fusion protein (His)(6)-UBI-CA-MA. The fusion protein was expressed in soluble form under the optimized conditions at high level (more than 36% of the total proteins). With (His)(6)-tag, the fusion protein was easily purified by Ni-NTA chromatography and 36 mg of fusion protein was purified from 1L of culture medium. The fusion protein was efficiently cleaved by ubiquitin C-terminal hydrolase (UCH), yielding recombinant CA-MA with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant CA-MA was purified to homogeneity by reversed-phase HPLC and 6.8mg of pure active CA-MA was obtained from 1L culture medium. Analysis of recombinant CA-MA by MALDI-TOF-MS showed that the molecular weight of the purified recombinant CA-MA was 2559Da, which perfectly matches the mass (2559Da) calculated from the amino acid sequence. Analysis of CA-MA by circular dichroism (CD) revealed that the secondary structures of CA-MA in water solution were 17.4% alpha-helix and 82.6% random coil but no beta-sheet. Our results demonstrated that functional CA-MA can be produced in sufficient quantities using the ubiquitin fusion technique. This is the first report on the heterologous expression of a hybrid antibacterial peptide fused to ubiquitin in E. coli.
authors.affiliation: State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, PR China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Xu
authors.forename: Fengliang
authors.initials: F
authors.lastname: Jin
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Yu
authors.forename: Shunxiang
authors.initials: S
authors.lastname: Ren
authors.forename: Jian
authors.initials: J
authors.lastname: Hu
authors.forename: Wenqing
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D001419:Bacteria / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006793:Houseflies / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics; D043222:Ubiquitin Thiolesterase / Q000737:chemistry
chemical_list: D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C121751:cecropin A(1-8)magainin 2(1-12); D043222:Ubiquitin Thiolesterase