Contribution of bovine lactoferrin inter-lobe region to iron binding stability and antimicrobial activity against Staphylococcus aureus

Abstract

The investigation of the recombinant bovine lactoferrin-derived antimicrobial protein (rBLfA) demonstrates that the inter-lobe region of bovine lactoferrin contributes to iron binding stability and antimicrobial activity against Staphylococcus aureus. rBLfA containing N-lobe (amino acid residues 1-333) and inter-lobe region (residues 334-344) was expressed in Pichia pastoris at shaking flask and fermentor level. The recombinant intact bovine lactoferrin (rBLf) and N-lobe (rBLfN) were expressed in the same system as control. The physical-chemical parameters of rBLfA, rBLfN and rBLf 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 rBLf and rBLfN. 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 6.5 micromol/L, compared with 12.5 and 25 micromol/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.

Functional analysis of recombinant buffalo lactoferrin and monoferric lobes and their cytotoxic effect on buffalo mammary epithelial cells

“…Iron binding to buLf C-lobe was comparatively much stronger. Similar iron release pattern has also been reported in case of recombinant bovine Lf (Bai et al 2010). The interactions in the hinge region of human Lf subdomains in the N and C-lobes indicated lower flexibility in C-lobe than in N-lobe.…”

“…The recombinant protein is shown to be indistinguishable from breast milk Lf by several criteria including iron and receptor binding (Bai et al 2010), antimicrobial activity (Ward et al 1997) and thermal stability (Chen et al 2007). In the present investigation we report (1) recombinant expression of buLf and its monoferric lobes, (2) characterization of iron-binding and biochemical properties of recombinant Lf vis-avis native milk Lf, (3) effect of recombinant Lf and its monoferric lobes on buffalo mammary epithelial cells (BuMEC) and casein expression and the possible mode of cytotoxic action of Lf against BuMEC.…”

Design and pharmacodynamics of recombinant NZ2114 histidine mutants with improved activity against methicillin-resistant Staphylococcus aureus

“…The pH was controlled at 5.5 using H 3 PO 4 and NH 4 OH, and the temperature was maintained at 29 °C. When the glucose was exhausted, methanol was supplied from 1 to 7 ml/l/h during the first 6 h. Then methanol was supplied to maintain a relative dissolved oxygen (DO) content between 20 and 40% under the speed of 6–8 ml/l/h (Bai et al 2010 ). The fermentation liquid was collected every 24 h to quantify the cell wet weight and the total protein level which was assayed by a Bradford protein assay kit (Tiangen Biotech, Beijing, China).…”

Biotechnical paving of recombinant enterocin A as the candidate of anti-Listeriaagent

“…The whole fermentation period was performed at 29°C. During the glucose batch and glucose-fed phases, the pH was kept at 5.0 and increased to 5.5 at the methanol induction phase [42]. The protein in the supernatant was determined by the Bradford protein assay (Tiangen, Beijing, China) and Tricine-SDS–PAGE [43].…”