Molecular Design And Structure - Activity Relationship Of Bovine Lactoferrin Derived Fregments

Bovine lactoferrin (BLf) is a non-heme iron binding glycoprotein being considered as the first line defense protein involved in protection against microbial infection and prevention of systemic inflammation. The contribution of bovine lactoferrin (BLf) inter-lobe region to iron binding stability and antimicrobial activity against Staphylococcus aureus were addressed in this work.A recombinant bovine lactoferrin-derived antimicrobial protein (rBLfA) containing N-lobe (amino acid residues 1-333) and inter lobe region (residues 334-344) of bovine lactoferrin (BLf) was designed based on the structural and functional analysis of BLf. Recombinant full length BLf (rBLf), the N-lobe (rBLfN) without the inter lobe region and the N-lobe-derived peptide rBLfB (residues 1-284) were also designed following the same methords as control. The physical-chemical parameters of BLfA, BLfB, BLfN and BLf including amino acid residues, molecular weight, isoelectric point, net positive charge and instability index were computed and compared. The simulated tertiary structure and the calculated surface net charge showed that rBLfA maintained original structure and exhibited a higher cationic feature than BLfB, BLfN and BLf.The full length cDNA (2067 bp) of BLf and the coding gene of BLfA, BLfB and BLfN were cloned from the bovine mammary tissue. BLfA, BLfB and BLf were expressed in E. coli with the yields of 56, 41 and 96 mg/L, respectively. BLfA, BLfB, BLfN and BLf were all secretory expressed in the methylotrophic yeast Pichia pastoris. The yield of rBLfA in shaking flask culture was over 485 mg/L, which was 4.6, 8.7 and 5.5 times bigger than the yields of rBLfB, rBLfN and rBLf, respectively. Purified rBLfA, rBLfB, rBLfN and rBLf were obtained via Ni-IDA His-bind columns with the purity of 93.8%,92.0%,94.5% and 91.2%.The iron binding and antimicrobial activities comparison between rBLfA, rBLfN and rBLf demonstrates that the inter-lobe region of bovine lactoferrin contributes to iron binding stability and antimicrobial activity against S. aureus. The three proteins showed different iron binding stability and antimicrobial activity. rBLfA released iron in the pH range of 7.0-3.5, whereas rBLfN lost its iron over the pH range of 7.0-4.0 and iron release from rBLf occurred in the pH range of 5.5-3.0. However, the minimum inhibition concentration of rBLfA against S. aureus ATCC25923 was 6.5μmol/L, compared with 12.5μmol/L and 25μmol/L that of rBLfN and rBLf, respectively. These results revealed that S. aureus was more sensitive to rBLfA than rBLfN and rBLf. It appeared that the strong cationic character of inter-lobe region related positively to the higher anti-S. aureus activity.High density fermentation and Vitreoscilla hemoglobin (VHb) intracellular co-expression were employed to increase the in vitro expression yield of rBLfA. After 72 h methanol induction, the yield of rBLfA reached 613 mg/L by the high density fermentation of recombinant P. pastoris X-33(rBLfA-VHb).