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http://dx.doi.org/10.4014/jmb.0907.07029

Simple Purification of the Human Antimicrobial Peptide Dermcidin (MDCD-1L) by Intein-Mediated Expression in E. coli  

Hong, In-Pyo (Department of Bioengineering and Technology, Kangwon National University)
Kim, Yong-Seok (Department of Biochemistry, College of Medicine, Hanyang University)
Choi, Shin-Geon (Department of Bioengineering and Technology, Kangwon National University)
Publication Information
Journal of Microbiology and Biotechnology / v.20, no.2, 2010 , pp. 350-355 More about this Journal
Abstract
Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.
Keywords
Antimicrobial peptides; DCD-1L; dermcidin; intein; protein expression;
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1 Xu, M.-Q. and T. C. Evans Jr. 2005. Recent advances in protein splicing: Manipulating proteins in vitro and in vivo. Curr. Opin. Biotech. 16: 440-446.   DOI   ScienceOn
2 Hong, I. P., Y. S. Kim, and S. G. Choi. 2007. Recombinant expression of human cathelicidin (hCAP18/Ll37) in Pichia pastoris. Biotechnol. Lett. 29: 73-78.
3 Lai, Y. P., Y. F. Peng, Y. Zuo, J. Li, J. Huang, L. F. Wang, and Z. R. Wu. 2005. Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide. Biochem. Biophys. Res. Commun. 328: 243-250.   DOI   ScienceOn
4 Sambrook, J. and D. W. Russell. 2000. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.
5 Sitaram, N. and R. Nagaraj. 1999. Interaction of antimicrobial peptides with biological and model membranes: Structural and charge requirements for activity. Biochim. Biophys. Acta 1462: 29-54.   DOI   ScienceOn
6 Morassutti, C., F. De Amicis, A. Bandiera, and S. Marchetti. 2005. Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system. Protein Expr. Purif. 39: 160-168.   DOI   ScienceOn
7 Schroder, J. M. and J. Harder. 2006. Antimicrobial skin peptides and proteins. Cell Mol. Life Sci. 63: 469-486.   DOI   ScienceOn
8 Chen, Y. Q., S. Q. Zhang, B. C. Li, W. Qiu, B. Jiao, J. Zhang, and Z. Y. Diao. 2008. Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners. Protein Expr. Purif. 57: 303-311.   DOI   ScienceOn
9 Flad, T., R. Bogumil, J. Tolson, B. Schittek, C. Garbe, M. Deeg, C. A. Mueller, and H. Kalbacher. 2002. Detection of dermcidinderived peptides in sweat by ProteinChip technology. J. Immunol. Methods 270: 53-62.   DOI   ScienceOn
10 Steffen, H., S. Rieg, I. Wiedemann, H. Kalbacher, M. Deeg, H. G. Sahl, et al. 2006. Naturally processed dermcidin-derived peptides do not permeabilize bacterial membranes and kill microorganisms irrespective of their charge. Antimicrob. Agents Chemother. 50: 2608-2620.   DOI   ScienceOn
11 Baechle, D., T. Flad, A. Cansier, H. Steffen, B. Schittek, J. Tolson, et al. 2006. Cathepsin D is present in human eccrine sweat and involved in the postsecretory processing of the antimicrobial peptide DCD-1L. J. Biol. Chem. 281: 5406-5415.
12 Huang, L., J. Wang, Z. Zhong, L. Peng, H. Chen, Z. Xu, and P. Chen. 2006. Production of bioactive human beta-defensin-3 in Escherichia coli by soluble fusion expression. Biotechnol. Lett. 28: 627-632.   DOI   ScienceOn
13 Ingham, A. B. and R. J. Moore. 2007. Recombinant production of antimicrobial peptides in heterologous microbial systems. Biotechnol. Appl. Biochem. 47: 1-9.   DOI   ScienceOn
14 Rieg, S., C. Garbe, B. Sauer, H. Kalbacher, and B. Schittek. 2004. Dermcidin is constitutively produced by eccrine sweat glands and is not induced in epidermal cells under inflammatory skin conditions. Br. J. Dermatol. 151: 534-539.   DOI   ScienceOn
15 Senyurek, I., M. Paulmann, T. Sinnberg, H. Kalbacher, M. Deeg, T. Gutsmann, et al. 2009. Dermcidin-derived peptides show a different mode of action than the cathelicidin LL-37 against Staphylococcus aureus. Antimicrob. Agents Chemother. 53: 2499-2509.   DOI   ScienceOn
16 Reddy, K. V., R. D. Yedery, and C. Aranha. 2004. Antimicrobial peptides: Premises and promises. Int. J. Antimicrob. Agents 24: 536-547.   DOI   ScienceOn
17 Diao, H., C. Guo, D. Lin, and Y. Zhang. 2007. Intein-mediated expression is an effective approach in the study of betadefensins. Biochem. Biophys. Res. Commun. 357: 840-846.   DOI   ScienceOn
18 Bisht, G. S., D. S. Rawat, A. Kumar, R. Kumar, and S. Pasha. 2007. Antimicrobial activity of rationally designed amino terminal modified peptides. Bioorg. Med. Chem. Lett. 17: 4343-4346.   DOI   ScienceOn
19 Cipakova, I., J. Gasperik, and E. Hostinova. 2006. Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli. Protein Expr. Purif. 45: 269-274.   DOI   ScienceOn
20 Schittek, B., R. Hipfel, B. Sauer, J. Bauer, H. Kalbacher, S. Stevanovic, et al. 2001. Dermcidin: A novel human antibiotic peptide secreted by sweat glands. Nat. Immunol. 2: 1133-1137.   DOI   ScienceOn