Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 55, Issue 1, Pages 175-182Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2007.04.018
Keywords
hybrid peptide CA-MA; ubiquitin; fusion expression; purification; anti-microbial activity
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The hybrid antibacterial peptide CA-MA [ceeropinA(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 I 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 I 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% alpha-helix and 82.6% random coil but no P-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 eoli. (C) 2007 Elsevier lnc. All rights reserved.
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