Abstract
Antimicrobial peptide CM4 is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells. Different strategies have been developed to produce small antibacterial peptides using recombinant techniques. To date, no efforts to obtain large quantities of active recombinant CM4 have been reported. In order to establish a bacterium-based CM4 production system, CM4 was cloned into pET28a and expressed with Npro mutant (EDDIE) fusion. CM4 expressed as EDDIE are deposited as inclusion bodies. On in vitro refolding by switching from chemotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving CM4 protein with an authentic N terminus. Purified CM4 was separated on Ni2+-chelating chromatography column and cation-exchange chromatography column. Mass spectroscopic analysis indicated the protein to be 4132.56 Dalton, which equalled the theoretically expected mass. N-terminal sequencing of CM4 showed the sequence corresponded to the native protein. The recombinant CM4 exhibited the same antimicrobial and anti-tumor activity as reported previously. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant CM4 with native sequences.
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Acknowledgments
We thank Dr. Bernhard Auer, Institute of Biochemistry, University of Innsbruck, Austria, for kindly providing us with vector pET30/EDDIE. This research was supported by the grant from Jiangsu Province’s Outstanding Leader Program of Traditional Chinese Medicine.
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Cheng, X., Lu, W., Zhang, S. et al. Expression and purification of antimicrobial peptide CM4 by Npro fusion technology in E. coli . Amino Acids 39, 1545–1552 (2010). https://doi.org/10.1007/s00726-010-0625-0
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DOI: https://doi.org/10.1007/s00726-010-0625-0