Design, expression and characterization of recombinant hybrid peptide Attacin-Thanatin in Escherichia coli
- 223 Downloads
Antimicrobial peptides will be attractive and potential candidates as peptide drugs because of their efficient action against microbes and low toxicity to mammal cells. To improve their antibacterial activity, some modifications needs to be made. In this research, the hybrid peptide gene Attacin-Thanatin with 642 bp in length with preferred codons of E. coli was generated using the technology of Gene splicing by overlap extension. The gene was inserted in-frame into E. coli expression plasmid pET-32a (+) and induced to express in E. coli Rosetta. The recombinant protein was partial purified and its biological activity was determined. Analysis of the E. coli Rosetta induced with IPTG revealed that the molecular weight of fusion protein was approximately 41.8 kDa, which perfectly matched the mass calculated from the amino acid sequence. Biological activity detection showed that this peptide effectively inhibited the growth of the test bacteria including E. coli DH5α, E. coli BL21 (DE3), Salmonella choleraesuis and Staphylococcus aureus. Among these bacteria, the Gram-negative E. coli was the most sensitive. Furthermore, there was minor hemolysis activity for porcine red blood cells. So, the results indicated that the hybrid peptide Attacin-Thanatin could be served as a promising candidate for the chemical antibiotics.
KeywordsAntimicrobial peptides Hybrid peptide Attacin A Thanatin Prokaryotic expression Antimicrobial activity
The authors thank Mr. X. F. Liu for providing the agarose beads used for protein purification. Project was funded by grants from Program for Changjiang Scholars and Innovative Research Team in University with grant. No. IRTO 555-5, China Ministry of Education, and Feed Biotechnology Project of Sichuan Province of China with grant No.2007Z06-050.
- 3.Hultmark D, Engström A, Andersson K, Steiner H, Bennich H, Boman HG (1983) Insect immunity: attacins, a family of antibacterial proteins from Hyalophora cecropia. EMBO 2:571–576Google Scholar
- 7.Sugiyama M, uniyoshi HK, Kotani E, Taniai K, Kadono-Okuda K, Kato Y, Yamamoto M, Shimabukuro M, Chowdhury S, Xu J, Choi SK, Kataoka H, Suzuki A, Yamakawa M (1995) Characterization of a Bombyx mori cDNA encoding a novel member of the attacin family of insect antibacterial proteins. Insect Biochem Mol Biol 25:385–392CrossRefPubMedGoogle Scholar
- 8.Zhang B, Wang LM, Ye B, Li SY, Zhao ZJ, Fan Q (2006) Cloning and analysis of cDNA of the attacin gene from the Chinese oak silkworm, Antheraea pernyi, using RLM-RACE method. Canye Kexue 32:333–339Google Scholar
- 9.Hu YJ, Aksoy S (2002) In vitro expression of tsetse attacin and its activity against Trypanosoma brucei. Am J Trop Med Hyg 67:371Google Scholar
- 10.Xu JH, Zhu JY, Jin XB, Xu QY (2007) Cloning and expression of antibacterial peptide Attacin gene isolated from Musca Domestica Larvae and its biological activities. J Med Mol Biol 4:20–26Google Scholar
- 12.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 93:1221–1225CrossRefPubMedGoogle Scholar
- 18.Wingfield PT. (2005) Preparation of soluble proteins from Escherichia coli. Curr Protoc Protein Sci. Chapter 6 Unit 6. 2Google Scholar
- 20.Wu M, Hancock RE (1999) Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J Biol Chem 274:28–35Google Scholar
- 30.Giacometti A, Cirioni O, Kamysz W, D’Amato G, Silvestri C, Prete MSD et al (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 Microb Infect Dis 49:197–200CrossRefGoogle Scholar
- 31.Giacometti A, Cirioni O, Kamysz W, D’Amato G, Silvestri C, Prete MSD et al (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 24:1315–1318CrossRefPubMedGoogle Scholar