Abstract
With great therapeutic potential against antibiotic-resistant bacteria, viruses, and even parasites, antimicrobial peptides (AMPs) have received increased interest as pharmaceutical agents in recent years. It is a worthy yet challenging work to carry out the implement and improvement of AMPs production using bioengineering techniques. In the present study, a novel hybrid peptide LFT33 was designed derived from LfcinB and thanatin. The cDNA fragment encoding LFT33 with preferred codons of Escherichia coli was chemically synthesized and ligated into the vector pET32a(+) to express the LFT33 fusion protein. The fusion protein was successfully expressed in soluble form in E. coli induced under optimized conditions. After purification by affinity chromatography, the fusion protein was cleaved successfully by enterokinase and released the peptide LFT33. About 0.5 mg of the recombinant LFT33 was obtained by reversed-phase high performance liquid chromatography from 1 l of culture medium. Mass spectrometry analysis of the purified recombinant LFT33 demonstrated that the molecular weight perfectly matched the calculated mass (4,195 Da). The recombinant peptide LFT33 caused an increase in antimicrobial activity (IC50 = 16–64 μg/ml) against given strains and did not show hemolytic activity for human erythrocytes. The results indicated that the hybrid peptide LFT33 could serve as a promising candidate for pharmaceutical agents.
Similar content being viewed by others
References
Andreu D, Ubach J, Boman A, Wåhlin B, Wade D, Merrifield RB, Boman HG (1992) Shortened cecropin A-melittin hybrids. Significant size reduction retains potent antibiotic activity. FEBS Lett 296(2):190–194
Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M (1992) Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J Appl Bacteriol 73(6):472–479
Bellamy W, Wakabayashi H, Takase M, Kawase K, Shimamura S, Tomita M (1993) Killing of Candida albicans by lactoferricinB, a potent antimicrobial peptide derived from the N-terminal region of bovine lactoferrin. Med Microbiol Immunol (Berl) 182(2):97–105
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
Dathe M, Wieprecht T (1999) Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes. Biochim Biophys Acta 1462(1–2):71–87
Diamond G, Beckloff N, Weinberg A, Kisich KO (2009) The roles of antimicrobial peptides in innate host defense. Curr Pharm Des 15(21):2377–2392
Fehlbaum P, Bulet P, Chernysh S, Briand JP, Roussel JP, Letellier L, Hetru C, Hoffmann JA (1996) Structure–activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides. Proc Natl Acad Sci USA 93(3):1221–1225
Ferre R, Melo MN, Correia AD, Feliu L, Bardají E, Planas M, Castanho M (2009) Synergistic effects of the membrane actions of cecropin–melittin antimicrobial hybrid peptide BP100. Biophys J 96(5):1815–1827
Freiburghaus C, Janicke B, Lindmark-Månsson H, Oredsson SM, Paulsson MA (2009) Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line. J Dairy Sci 92(6):2477–2484
Gobbo M, Benincasa M, Bertoloni G, Biondi B, Dosselli R, Papini E, Reddi E, Rocchi R, Tavano R, Gennaro R (2009) Substitution of the arginine/leucine residues in apidaecin Ib with peptoid residues: effect on antimicrobial activity, cellular uptake, and proteolytic degradation. J Med Chem 52(16):5197–5206
Hancock RE, Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24(12):1551–1557
Haversen L, Ohlsson BG, Hahn-Zoric M, Hanson LA, Mattsby-Baltzer I (2002) Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell Immunol 220(2):83–95
Huang L, Leong SS, Jiang R (2009) Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27. Appl Microbiol Biotechnol 84(2):301–308
Ingham AB, Moore RJ (2007) Recombinant production of antimicrobial peptides in heterologous microbial systems. Biotechnol Appl Biochem 47(Pt 1):1–9
Kim JM, Jang SA, Yu BJ, Sung BH, Cho JH, Kim SC (2008) High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage. Appl Microbiol Biotechnol 78(1):123–130
Koo YS, Kim JM, Park IY, Yu BJ, Jang SA, Kim KS, Park CB, Cho JH, Kim SC (2008) Structure–activity relations of parasin I, a histone H2A-derived antimicrobial peptide. Peptides 29(7):1102–1108
Lee MK, Cha L, Lee SH, Hahm KS (2002) Role of amino acid residues within the disulfide loop of thanatin, a potent antibiotic peptide. J Biochem Mol Biol 35(3):291–296
Li L, Wang JX, Zhao XF, Kang CJ, Liu N, Xiang JH, Li FH, Sueda S, Kondo H (2005) High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris. Protein Expr Purif 39(2):144–151
Maher S, McClean S (2006) Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro. Biochem Pharmacol 71(9):1289–1298
Marr AK, Jenssen H, Moniri MR, Hancock RE, Panté N (2009) Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1. Biochimie 91(1):160–164
Monincová L, Budesínský M, Slaninová J, Hovorka O, Cvacka J, Voburka Z, Fucík V, Borovicková L, Bednárová L, Straka J, Cerovský V (2010) Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs. Amino Acids 39(3):763–775
National Committee for Clinical Laboratory Standards (2003) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard M7-A6. Villanova, PA
Omata Y, Satake M, Maeda R, Saito A, Shimazaki K, Yamauchi K, Uzuka Y, Tanabe S, Sarashina T, Mikami T (2001) Reduction of the infectivity of Toxoplasma gondii and Eimeria stiedai sporozoite by treatment with bovine lactoferricin. J Vet Med Sci 63:187–190
Oo TZ, Cole N, Garthwaite L, Willcox MD, Zhu H (2010) Evaluation of synergistic activity of bovine lactoferricin with antibiotics in corneal infection. J Antimicrob Chemother 65(6):1243–1251
Park Y, Lee DG, Hahm KS (2004) HP(2–9)-magainin 2(1–12), a synthetic hybrid peptide, exerts its antifungal effect on Candida albicans by damaging the plasma membrane. J Pept Sci 10(4):204–209
Rekdal Ø, Andersen J, Vorland LH, Svendsen JS (1999) Construction and synthesis of lactoferricin derivatives with enhanced antibacterial activity. J Pept Sci 5(1):32–45
Scudiero O, Galdiero S, Cantisani M, Di Noto R, Vitiello M, Galdiero M, Naclerio G, Cassiman JJ, Pedone C, Castaldo G, Salvatore F (2010) Novel synthetic, salt-resistant analogs of human beta-defensins 1 and 3 endowed with enhanced antimicrobial activity. Antimicrob Agents Chemother 54(6):2312–2322
Shin SY, Kang JH, Lee MK, Kim SY, Kim Y, Hahm KS (1998) Cecropin A-magainin 2 hybrid peptides having potent antimicrobial activity with low hemolytic effect. Biochem Mol Biol Int 44(6):1119–1126
Spellberg B, Guidos R, Gilbert D, Bradley J, Boucher HW, Scheld WM, Bartlett JG, Edwards J Jr (2008) The epidemic of antibiotic-resistant infections: a call to action for the medical community from the Infectious Diseases Society of America. Clin Infect Dis 46(2):155–164
Strøm MB, Rekdal Ø, Svendsen JS (2000) Antibacterial activity of 15-residue lactoferricin derivatives. J Pept Res 56(5):265–274
Strøm MB, Haug BE, Rekdal Ø, Skar ML, Stensen W, Svendsen JS (2002) Important structural features of 15-residue lactoferricin derivatives and methods for improvement of antimicrobial activity. Biochem Cell Biol 80(1):65–74
Taguchi S, Kuwasako K, Suenaga A, Okada M, Momose H (2000) Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system. J Biochem 128(5):745–754
Tian ZG, Dong TT, Teng D, Yang YL, Wang JH (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(6):1097–1103
Ulvatne H, Samuelsen Ø, Haukland HH, Krämer M, Vorland LH (2004) Lactoferricin B inhibits bacterial macromolecular synthesis in Escherichia coli and Bacillus subtilis. FEMS Microbiol Lett 237(2):377–384
Wakabayashi H, Teraguchi S, Tamura Y (2002) Increased Staphylococcus-killing activity of an antimicrobial peptide, lactoferricin B, with minocycline and monoacylglycerol. Biosci Biotechnol Biochem 66(10):2161–2167
Wu M, Hancock RE (1999) Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J Biol Chem 274(1):29–35
Zasloff M (2002) Antimicrobial peptides of multicellular organism. Nature 414(6870):389–395
Zorko M, Jerala R (2010) Production of recombinant antimicrobial peptides in bacteria. Meth Mol Biol 618:61–76
Acknowledgments
This work was supported by the grants from National Natural Science Foundation of China (30800794), Youth Science Funds of Heillongjiang Province (QC08C05), and Postdoctoral Research Initiation Founds of Heillongjiang Province (LBH-Q10145).
Author information
Authors and Affiliations
Corresponding author
Additional information
Xingjun Feng and Chunlong Liu contributed equally to this work.
Rights and permissions
About this article
Cite this article
Feng, X., Liu, C., Guo, J. et al. Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33. Appl Microbiol Biotechnol 95, 1191–1198 (2012). https://doi.org/10.1007/s00253-011-3816-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-011-3816-z