Cloning and expression of a gene encoding shortened LfcinB(1-15)-Melittin(5-12) hybrid peptide in Escherichia coli BL21(DE3).
- Author:
Chongpeng BI
1
;
Xingjun FENG
;
Anshan SHAN
;
Jiayin GUO
Author Information
1. Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Anti-Bacterial Agents;
biosynthesis;
Antimicrobial Cationic Peptides;
biosynthesis;
chemistry;
genetics;
Cattle;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
Lactoferrin;
biosynthesis;
genetics;
Melitten;
biosynthesis;
genetics;
Recombinant Fusion Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2009;25(7):975-981
- CountryChina
- Language:Chinese
-
Abstract:
In order to get new antibacterial peptide, we designed a hybrid peptide LfcinB(1-15)-Melittin(5-12), composed of 1-15 amino acid residues of bovine Lactoferricin and 5-12 amino acid residues of Melittin. According to the bias of codon utilization of Escherichia coli, We synthesized the gene encoding the hybrid peptide. We inserted the gene between the sites of Nco I and Sal I of pET-32a and obtained the recombinant expression vector for heterologous expression of LfcinB(1-15)-Melittin(5-12) in Escherichia coli. We used Escherichia coli BL21(DE3) as expression host for the recombinant plasmid. After induced by isopropyl-beta-D-thiogalactoside (IPTG) under the optimized conditions, we realized the fusion protein was successfully expressed. The fusion protein was expressed in soluble form and the level was more than 35% of the total proteins. With (His)6 x Tag, the fusion protein was easily purified by His x Bind Purification Kit. After purification, we obtained 35 mg of fusion protein from 1 L of culture medium. At last, we accomplished that the peptide LfcinB(1-15)-Melittin(5-12) was released from the fusion protein cleaved by enterokinase. The recombinant LfcinB(1-15)-Melittin(5-12) showed antimicrobial activity assayed by agar diffusion test. This is the first report on the heterologous expression of the hybrid antibacterial peptide LfcinB(1-15)-Melittin(5-12) in Escherichia coli and also provides basis for next cost-effective expression of other antimicrobial peptides in genetic engineering.
