Multimerization And Fusion Expression Of Antimicrobial Peptide LfcinB15-W4, 10

Such argue on the remnant of antibiotic and antibiotic-resistant strains were increasingly vigorous with the wide application of traditional antibiotics. The great concern to develop effective but human, animals and environmental compatible antibiotic alternatives is one of the focus researches. Antimicrobial peptide has great potential and can be applied in many fields as one of antibiotics alternative due to its advantage of possessing more effective activity as well as exhibiting unique mechanism against bacteria. Bovine lactoferricin (LfcinB) and its derivatives have efficient antimicrobial activity and other biological functions. LfcinB15-W4,10 is a cationic antimicrobial peptide composed of 15 amino acid residues derived from LfcinB. LfcinB15-W4,10 has more effective antimicrobial activity than native LfcinB because of modification of amino acid sequence, and don't have rare amino acids and heterogenous chemical compositions, so it is one kind of great potential and secure antibiotics alternative. In this study, antimicrobial activity of LfcinB15-W4,10 and antibiotics are compared. And LfcinB15-W4,10 was expressed successfully by gene engineering, which is beneficial for industrial purpose. The following results were obtained in this study.In the experiment of antimicrobial activity in vitro, LfcinB15-W4,10 chemically synthesized displayed less biological activity than penicillin, chlortetracycline, streptomycin and doxycycline, but LfcinB15-W4,10 showed the antimicrobial activity against test bacteria in deed. The gene encoding the peptide LfcinB15-W4,10 were synthesized based on the amino acid sequences according to the codon usage of E. coli, and multimerized by T4 DNA ligase with asymmetric cohesive ends 5'-CCGA/5'-TCGG The multimeric gene of LfcinB15-W4,10 was inserted into linearized pET32a by BamH â…  and fluid â…¢ adapter for construction of recombinant vector pET32a-(LfcinB15-W4,10)_n. The constructs containing peptide monomer to nonamer but octamer were confirmed by colony PCR and DNA sequencing. After IPTG induction, the result of SDS-PAGE gel indicates that the different fusion multimeric peptide were successfully induced and translated respectively. In which, the fused LfcinB15-W4,10 tetramer showed the highest expression, about 45% of total cell proteins. After Ni²⁺ chromatography purification, 73 mg fusion protein Trx-(LfcinB15-W4,10)₄ with purity of 86.3% was obtained from 1 litre of E. coli culture. The fusion tetramer was effectively cleaved by thrombin at a final concentration of 5 U/mg at 25 ℃ for 48 hrs. The tetramer with the purity of 99% was electro-eluted after cleavage, displayed antimicrobial activity against 5. aureus ATCC 25923. The MIC against S. aureus ATCC 25923 for recombinant tetramer was 16 μg/mL evaluated by the modified microtitre broth dilution method.The antimicrobial peptide LfcinB15-W4,10 derived from LfcinB was multimerized successfully and the fusion peptide was expressed with high level in E.coli BL21(DE3). 10 mg of tetramer with 99% purity was obtained from 1 litre culture and exhibited antimicrobial activity. The proposed expression system provides a potential production method for LfcinB15-W4,10 and other antimicrobial peptides to industrial and research application.