Expression Of Bovine Lactoferrin Gene Fragment In Prokaryotic Cells And The Activity Analysis Of Bovine Lactoferricin

Abstract:Antibiotic resistance has become a major concern for animal feed industry and human medicine worldwide. As a promising alternative of antibiotics, Bovine lactoferricin (LfcinB) is a peptide of 25 amino acids that originates from the N-terminus (Phel7 to Phe41) of bovine lactoferrin. Because LfcinB has broad spectrum of potent antimicrobial activity, it is highly toxic to heterologous expression host cells like E. coli cells. The objective of this experiment was to develop an efficient expression system to produce an inactive or non-toxic precursor of LfcinB in E. coli. We synthesized a DNA fragment encoding the N-terminal 121 amino acids of bovine lactoferrin that contained the LfcinB peptide, inserted the DNA fragment into an expression vector pET-30a(+), and delivered the construct into E. coli strain Rosetta (DE3) cells. As shown by the SDS-PAGE analysis, upon induction by IPTG, a truncated bovine lactoferrin with a molecular mass of approximately 19.2 kDa was produced by the transformants, and the yield accounted for-30% of the total cell protein. The protein purified by Ni-Sepharose affinity chromatography (GE Healthcare, Piscataway, NJ) exhibited a molecular mass of approximately 30 kDa as determined by the SDS-PAGE analysis. After that the antibacterial activity of Lfcin B released from the truncated was determined, recombinant protein by pepsin digestion. In summary, our approach in producing the active LfcinB precursor may provide an alternative way to produce LfcinB at low cost and convenience.