Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant baculovirus and its antimicrobial activity against Prototheca zopfii

Abstract

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in secretory fluids of mammals. In this study, DNA encoding bovine lactoferrin (bLF) or the N-terminal half of bLF (bLF N-lobe) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLF or bLF N-lobe was isolated. An 80-kDa bLF-related protein expressed by the recombinant baculovirus was detected by monoclonal antibodies against bLF N-lobe and the C-terminal half of bLF (bLF C-lobe). A 43-kDa bLF N-lobe-related protein expressed by the recombinant baculovirus was detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody. These proteins were also secreted into the supernatant of insect cell cultures. Recombinant bLF (rbLF) and bLF N-lobe (rbLF N-lobe) were affected by tunicamycin treatment, indicating that rbLF and rbLF N-lobe contain an N-linked glycosylation site. Antimicrobial activity of these recombinant proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis) was evaluated by measuring the optical density of the culture microplate. Prototheca zopfii was sensitive to rbLF and rbLF N-lobe, as well as native bLF. There was no difference in antimicrobial activity between rbLF N-lobe and bLF C-lobe.

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

“…The first two regions are both located at BLf N-lobe which contains functional domains (lactoferricin and lactoferrampin) associated with the bactericidal activity. In vitro expressions of recombinant BLf N-lobe have been developed for the structures, functions, and applications studies (Nakamura et al 2001;Tanaka et al 2003). However, investigations in the structural and functional properties of the inter-lobe region (residues 334-344) have not been closely concerned.…”

Expression and characterization of bovine lactoperoxidase by recombinant vaccinia virus

“…The sequence analysis was done using an Applied Biosystems sequencer (Foster City, CA, USA). bLF cDNA was amplified from mamary glands cells, using RT-PCR with primers designed from bLF cDNA (Tanaka et al 2003a). The PCR products were blunted by T4 DNA polymerase and ligated with the vaccinia virus transfer vector pAK8 (Yasuda et al 1990), which was cut with Sal I and then blunted.…”

Expression of Bovine Lactoferrin C-lobe inRhodococcus erythropolisand Its Purification and Characterization

“…Recently, Tanaka et al 20) and Nakamura et al 17) reported the expression of rBLF and the lactoferrin Nlobe using a Spodoptera frugiperda (Sf9) cell as the host cell. In these studies, the expression of the rBLF C-lobe did not succeed, but we successfully expressed and isolated substantial amounts of the BLF C-lobe using the expression system with an inducible expression vector.…”