Expression and characterization of recombinant bovine lactoferrin in E. coli

Abstract

Lactoferrin is a member of the transferrin family of iron-binding proteins with a number of properties, including antibacterial activity against a broad spectrum of Gram-negative and Gram-positive bacteria. bovine lactoferrin cDNA was isolated, cloned and expressed as a fusion protein. The amino acid sequence of the fusion was analyzed and compared with other species. Crystallographic data were used to compare structural differences between bovine and human lactoferrin in 3-D models. A thioredoxin fusion protein was expressed and shown to have a different molecular weight compared with native bLf. After purification using Ni-NTA, the yield of recombinant bovine lactoferrin was 15.3 mg/l with a purity of 90.3 %. Recombinant bLf and pepsin-digested rbLf peptides demonstrated antibacterial activity of 79.8 and 86.9 %, respectively. The successful expression of functional, active and intact rbLf allows us to study the biochemical interactions of antimicrobial proteins and peptides and will facilitate their study as immunomodulators.

High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

“…The synthetic bovine lactoferrin ( sbLf ) gene is derived from a bLf gene (GenBank accession No. EU812318) cloned by García-Montoya et al [ 15 ] and is under the control of the AOX1 promoter. Alkaline lysis extraction and digestion of the synthetic plasmid pJ902sbLf by BamHI produced a linearized DNA fragment that measured 5564 bp in length.…”

“…Currently, Gram-negative bacteria are becoming increasingly resistant to common antibiotics due to the presence of an outer membrane (OM) that has a unique structure creating an impermeable layer, which provides protection against antibiotic penetration [ 37 ]. In our last paper, we demonstrated that, unlike non-fragmented lactoferrin, pepsin-digested rbLf has a higher antibacterial potential against E. coli [ 15 ]. The proteolytic digestion of Lf by gastric pepsin releases a bioactive peptide of Lf called lactoferricin; because lactoferricin can alter the permeability of bacterial membrane, several studies support the idea that lactoferricin presents higher antibacterial activity that non-fragmented lactoferrin [ 38 ].…”

“…Lf has been expressed in several microscopic models. Our research group has expressed bovine lactoferrin (bLf) using an Escherichia coli ( E. coli ) model expression system, and we obtained functional antibacterial activity fractions of bLf [ 15 ]; however, the use of prokaryotic systems presents disadvantages which can be avoided by using eukaryotic systems—for example, the protein toxicity could interfere with growth and cause cell death [ 16 ].…”

Immunomodulatory effects of lactoferrin

“…It has been possible to produce recombinant Lf specific to human, bovine, equine, porcine, caprine, yak and Kunming by using various expression systems (eg, bacteria, fungi, yeast, cell lines, insects, mammals and plants). While Lf is produced in quantities ranging from 0.756 mg/L to 10.6 g/L, human Lf remains the most expressed among all of the different expression systems [87,[96][97][98][99][100][101][102][103] .…”

Lactobacillus pentosus expressing porcine lactoferrin elevates antibacterial activity and improves the efficacy of vaccination against Aujeszky’s disease

“…However, the traditional separation and purification of LF from the colostrum is a cumbersome process; therefore, the use of genetic engineering methods to produce recombinant LF has become a main focus of this technology (Wang et al, 1997;Wang et al, 2002;Pecorini et al, 2005;Chen et al, 2008). In genetically engineered cells, whether they are bacterial, yeast or animal cells, the expression and collection of exogenous protein usually require purification (Garcia-Montoya et al, 2013). The tedious purification process has been a barrier to applying genetically engineered cells in production.…”