Abstract
This study reports the first successful recombinant expression of cationic antimicrobial peptides human beta-defensin-26 and human beta-defensin-27 in Escherichia coli. HBD26 and HBD27 genes were synthesized through codon optimization, and each gene was then cloned into the expression vector pET32, which feature fusion protein thioredoxin at the N-terminal. The recombinant plasmids were then transformed into E. coli BL21 (DE3) and cultured in MBL medium, which gave yields of HBD26 and HBD27 fusion proteins of up to 1.38 and 1.29 g l−1, respectively. Affinity chromatography was used to purify the soluble fusion proteins, and the N-terminal TrxA tags were cleaved off by enterokinase. Pure HBD26 and HBD27 were then obtained by cationic exchange chromatography. The overall recovery of HBD26 was 38% and that of HBD27 reached 36%. Both variants showed salt-sensitive antimicrobial activity against gram-negative E. coli but not against gram-positive Staphylococcus aureus and Saccharomyces cerevisiae.
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References
Bensch KW, Raida M, Magert HJ, Schulzknappe P, Forssmann WG (1995) Hbd-1—a novel beta-defensin from human plasma. Febs Lett 368:331–335
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Epand RM, Vogel HJ (1999) Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta-Biomembr 1462:11–28
Ganz T (1999) Immunology—defensins and host defense. Science 286:420–421
Ganz T, Selsted ME, Szklarek D, Harwig SSL, Daher K, Bainton DF, Lehrer RI (1985) Defensins—natural peptide antibiotics of human-neutrophils. J Clin Invest 76:1427–1435
Garcia JRC, Krause A, Schulz S, Rodriguez-Jimenez FJ, Kluver E, Adermann K, Forssmann U, Frimpong-Boateng A, Bals R, Forssmann WG (2001) Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity. Faseb J 15:1819
Hannig G, Makrides SC (1998) Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotech 16:54–60
Harder J, Bartels J, Christophers E, Schroder JM (1997) A peptide antibiotic from human skin. Nature 387:861–861
Harder J, Bartels J, Christophers E, Schroder JM (2001) Isolation and characterization of human beta-defensin-3, a novel human inducible peptide antibiotic. J Biol Chem 276:5707–5713
Huang L, Ching CB, Jiang RR, Leong SSJ (2008) Production of bioactive human beta-defensin 5 and 6 in Escherichia coli by soluble fusion expression. Protein Expr Purif 61:168–174
Huang L, Wang JF, Zhong ZX, Peng L, Chen HQ, Xu ZN, Cen PL (2006) Production of bioactive human beta-defensin-3 in Escherichia coli by soluble fusion expression. Biotechnol Lett 28:627–632
Jia HP, Schutte BC, Schudy A, Linzmeier R, Guthmiller JM, Johnson GK, Tack BF, Mitros JP, Rosenthal A, Ganz T, McCray PB (2001) Discovery of new human beta-defensins using a genomics-based approach. Gene 263:211–218
Johansson AS, Bolton-Grob R, Mannervik B (1999) Use of silent mutations in cDNA encoding human glutathione transferase M2-2 for optimized expression in Escherichia coli. Protein Expr Purif 17:105–112
Jones DE, Bevins CL (1992) Paneth cells of the human small-intestine express an antimicrobial peptide gene. J Biol Chem 267:23216–23225
Jones DE, Bevins CL (1993) Defensin-6 messenger-Rna in human paneth cells—implications for antimicrobial peptides in host defense of the human bowel. Febs Lett 315:187–192
Lavallie ER, Diblasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the Escherichia-coli cytoplasm. Bio-Technology 11:187–193
Peng L, Xu ZN, Fang XM, Wang F, Yang S, Cen PL (2004) Preferential codons enhancing the expression level of human beta-defensin-2 in recombinant Escherichia coli. Protein Pept Lett 11:339–344
Piers KL, Brown MH, Hancock REW (1993) Recombinant-DNA procedures for producing small antimicrobial cationic peptides in bacteria. Gene 134:7–13
Rodriguez-Jimenez FJ, Krause A, Schulz S, Forssmann WG, Conejo-Garcia JR, Schreeb R, Motzkus D (2003) Distribution of new human beta-defensin genes clustered on chromosome 20 in functionally different segments of epididymis. Genomics 81:175–183
Schroder JM (1999) Epithelial antimicrobial peptides: innate local host response elements. Cell Mol Life Sci 56:32–46
Schutte BC, Mitros JP, Bartlettt JA, Walters JD, Jia HP, Welsh MJ, Casavant TL, McCray PB (2002) Discovery of five conserved beta-defensin gene clusters using a computational search strategy. Proc Natl Acad Sci U S A 99:2129–2133
Singh A, Bateman A, Zhu QZ, Shimasaki S, Esch F, Solomon S (1988) Structure of a novel human granulocyte peptide with anti-acth activity. Biochem Biophys Res Commun 155:524–529
Stewart EJ, Aslund F, Beckwith J (1998) Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. Embo J 17:5543–5550
Tang YQ, Yuan J, Osapay G, Osapay K, Tran D, Miller CJ, Ouellette AJ, Selsted ME (1999) A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science 286:498–502
Wilde CG, Griffith JE, Marra MN, Snable JL, Scott RW (1989) Purification and characterization of human neutrophil peptide-4, a novel member of the defensin family. J Biol Chem 264:11200–11203
Wong JH, Xia LX, Ng TB (2007) A review of defensins of diverse origins. Current Protein & Peptide Science 8:446–459
Yamaguchi Y, Nagase T, Makita R, Fukuhara S, Tomita T, Tominaga T, Kurihara H, Ouchi Y (2002) Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice. J Immunol 169:2516–2523
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Professor Chi Bun Ching (Nanyang Technological University, Singapore) is greatly acknowledged for provision of the strains used in the antimicrobial assays.
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Huang, L., Leong, S.S.J. & Jiang, R. Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27. Appl Microbiol Biotechnol 84, 301–308 (2009). https://doi.org/10.1007/s00253-009-1982-z
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DOI: https://doi.org/10.1007/s00253-009-1982-z