High-level expression of the recombinant hybrid peptide cecropinA(1-8)–magainin2(1-12) with an ubiquitin fusion partner in Escherichia coli

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 1 L 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 6.8 mg of pure active CA–MA was obtained from 1 L culture medium. Analysis of recombinant CA–MA by MALDI-TOF-MS showed that the molecular weight of the purified recombinant CA–MA was 2559 Da, which perfectly matches the mass (2559 Da) 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% α-helix and 82.6% random coil but no β-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.