The increasing problem of antibiotic resistance among pathogenic bacteria requires novel strategies for the construction of multiple, joined genes of antimicrobial agents. The strategy used in this study involved synthesis of a cDNA-encoding hinnavin II/α-melanocyte-stimulating hormone (hin/MSH) hybrid peptide, which was cloned into the pET32a (+) vector to allow expression of the hybrid peptide as a fusion protein in Escherichia coli BL21 (DE3). The resulting expression of fusion protein Trx-hin/MSH could reach up to 20% of the total cell proteins. More than 50% of the target protein was in a soluble form. The target fusion protein from the soluble fraction, Trx-hin/MSH, was easily purified by Ni2+-chelating chromatography. Then, enterokinase cleavage effectively cleaved the Trx-hin/MSH to release the recombinant hin/MSH (rhin/MSH) hybrid peptide. After removing the contaminants, we purified the recombinant hybrid peptide to homogeneity by reversed-phase FPLC and obtained 210 mg of pure, active rhin/MSH from 800 ml of culture medium. Antimicrobial activity assay demonstrated that rhin/MSH had a broader spectrum of activity than did the parental hinnavin II or MSH against fungi and Gram-positive and Gram-negative bacteria. These results suggest an efficient method for producing high-level expression of various kinds of antimicrobial peptides that are toxic to the host, a reliable and simple method for producing different hybrid peptides for biological studies.
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Andreu, D., J. Ubach, A. Boman, B. Wahlin, D. Wade, R.B. Merrifield, and H.G. Boman. 1992. Shortened cecropin A-melittin hybrids. Significant size reduction retains potent antibiotic activity. FEBS Lett. 296, 190–194.
Bao, W.J., Y.G. Gao, Y.G. Chang, T.Y. Zhang, X.J. Lin, X.Z. Yan, and H.Y. Hu. 2006. Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization. Protein Expr. Purif. 47, 599–606.
Boman, H.G., D. Wade, I.A. Boman, B. Wåhlin, and R.B. Merrifield. 1989. Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids. FEBS Lett. 259, 103–106.
Bradford, M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254.
Catania, A. and J.M. Lipton. 1993. α-Melanocyte stimulating hormone in the modulation of host reactions. Endor. Rev. 14, 564–576.
Cutuli, M., S. Cristianl, J.M. Lipton, and A. Catania. 2000. Antimicrobial effects of α-MSH peptides. J. Leukocyte Biol. 67, 233–239.
Ferre, R., E. Badosa, L. Feliu, M. Planas, E. Montesinos, and E. Bardají. 2006. Inhibition of plant-pathogenic bacteria by short synthetic cecropin A-melittin hybrid peptides. Appl. Environ. Microbiol. 72, 3302–3308.
Fink, J., R.B. Merrifield, A. Boman, and H.G. Boman. 1989. The chemical synthesis of cecropin D and an analog with enhanced antibacterial activity. J. Biol. Chem. 264, 6260–6267.
Giacometti, A., O. Cirioni, W. Kamysz, G.D Amato, C. Silvestri, M.S. Prete, J. Gukasiak, and G. Scalise. 2003. Comparative activities of cecropin A, melittin, and cecropin A-melittin peptide CA (1–7) M (2–9) NH2 against multidrug-resistant nosocomial isolates of Acinetobacter baumannii. Peptides 24, 1315–1318.
Giacometti, A., O. Cirioni, W. Kamysz, G.D Amato, C. Silvestri, M.S. Prete, J. Gukasiak, and G. Scalise. 2004. In vitro activity and killing effect of the synthetic hybrid cecropin A-melittin peptide CA (1–7) M (2–9) NH2 on methicillin-resistant nosocomial isolates of Staphylococcus aureus and interactions with clinically used antibiotics. Diagn. Microbiol. Infect Dis. 49, 197–200.
Hancock, R.E. 1997. Peptide antibiotics. Lancet 349, 418–422.
Hidari, K.I., N. Horie, T. Murata, D. Miyamoto, T. Suzuki, T. Usui, and Y. Suzuki. 2005. Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli. Glycoconj. J. 22, 1–11.
Ingham, A.B. and R.J. Moore. 2007. Recombinant production of antimicrobial peptides in heterologous microbial systems. Biotechnol. Appl. Biochem. 47, 1–9.
Jenssen, H., P. Hamill, and R.E.W. Hancock. 2006. Peptide antimicrobial agents. Clin. Microbiol. Rev. 19, 491–511.
Kim, J.M., S.A. Jang, B.J. Yu, B.H. Sung, J.H. Cho, and S.C. Kim. 2008. High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage. Appl. Microbiol. Biotechnol. 78, 123–130.
Kwak, H.B., S.W. Lee, D.G. Lee, K.S. Hahm, K.K. Kim, H.H. Kim, and Z.H. Lee. 2003. A hybrid peptide derived from cecropin-A and magainin-2 inhibits osteoclast differentiation. Life Sci. 73, 993–1005.
Laemmli, U.K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
LaVallie, E.R., E.A. DiBlasio, S. Kovacic, K.L. Grant, P.F. Schendel, and J.M. McCoy. 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology 11, 187–193.
Lee, D.G., Y. Park, P.I. Kim, H.G. Jeong, E.R. Woo, and K.S. Hahm. 2002. Infuence on the plasma membrane of Candida albicans by HP (2–9)-magainin 2 (1–12) hybrid peptide. Biochem. Biophys. Res. Commun. 297, 885–889.
Lipton, J.M. and A. Catania. 1997. Anti-inflamatory action of the neuroimmunomodulator α-MSH. Immunol. Today 18, 140–145.
Pazgier, M. and J. Lubkowski. 2006. Expression and purification of recombinant human a-defensins in Escherichia coli. Protein Expr. Purif. 49, 1–8.
Rao, X.C., S. Li, J.C. Hu, X.L. Jin, X.M. Hu, J.J. Huang, Z.J. Chen, J.M. Zhu, and F.Q. Hu. 2004. A novel carrier molecule for highlevel expression of peptide antibiotics in Escherichia coli. Protein Expr. Purif. 36, 11–18.
Sato, H. and J.B. Feix. 2008. Lysine-enriched cecropin-mellitin antimicrobial peptides with enhanced selectivity. Antimicrob. Agents Chemother. 52, 4463–4465.
Saugar, J.M., M.J. Rodríguez-Hernández, B.G. de la Torre, M.E. Pachón-Ibañez, M. Fernández-Reyes, D. Andreu, J. Pachón, and L. Rivas. 2006. Activity of cecropin A-melittin hybrid peptides against colistinresistant clinical strains of Acinetobacter baumannii: molecular basis for the differential mechanisms of action. Antimicrob. Agents Chemother. 50, 1251–1256.
Schägger, H. and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.
Sin, Y.S., H.J. Kang, Y.S. Jang, Y. Kim, L.K. Kim, and K.S. Hahm. 2000. Effects of the hinge region of cecropin A (1–8)-magainin 2 (1–12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells. Biochim. Biophys. Acta 1463, 209–218.
Skosyrev, V.S., E.A. Kulesskiy, A.V. Yakhnim, Y.V. Temirov, and L.M. Vinokurov. 2003. Expression of the recombinant antibacterial peptide sarcotoxin IA in Eschericha coli cells. Protein Expr. Purif. 28, 350–356.
Symersky, J., J. Novak, D.T. Mcpherson, L. Delucas, and J. Mestecky. 2000. Expression of the recombinant human immunoglobulin J chain in Escherichia coli. Mol. Immunol. 37, 133–140.
Tenno, T., N. Goda, Y. Tateishi, H. Tochio, M. Mishima, H. Hayashi, M, Shirakawa, and H. Hiroaki. 2004. High throughput construction, method for expression vector of peptides for NMR study suited for isotopic labeling. Protein Eng. Des. Sel. 174, 305–314.
Tian, Z.G., T.T. Dong, D. Teng, Y.L. Yang, and J.H. Wang. 2009. Design and characterization of novel hybrid peptides from LFB15 (W4, 10), HP (2–20), and cecropin A based on structure parameters by computer-aided method. Appl. Microbiol. Biotechnol. 82, 1097–1103.
Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.
Xu, X., F. Jin, X. Yu, S. Ren, J. Hu, and W. Zhang. 2007. High-level expression of the recombinant hybrid peptide cecropin A (1–8)-magainin 2 (1–12) with an ubiquitin fusion partner in Escherichia coli. Protein Expr. Purif. 55, 175–182.
Yoe, S.M., C.S. Kang, S.S. Han, and I.S. Bang. 2006. Characterization and cDNA cloning of hinnavinII, a cecropin family antibacterial peptide from the cabbage butterfly, Artogeia rapae. Comp. Biochem. Physiol. B. 144, 199–205.
Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415, 389–395.
Zhou, Q.F., X.G. Luo, L. Ye, and T. Xi. 2007. High-level production of a novel antimicrobial peptide perinerin in Escherichia coli by fusion expression. Curr. Microbiol. 54, 366–370.
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Bang, S.K., Kang, C.S., Han, MD. et al. Expression of recombinant hybrid peptide hinnavin II/α-melanocyte-stimulating hormone in Escherichia coli: Purification and characterization. J Microbiol. 48, 24–29 (2010). https://doi.org/10.1007/s12275-009-0317-1
- antimicrobial peptide
- α-melanocyte stimulating hormone (MSH)
- hybrid peptide
- fusion expression