Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity.
Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.
Nakamura
I
I
Dairy Science Laboratory, Faculty of Agriculture, Hokkaido University, N9W9 Kita-Ku, Sapporo, Hokkaido 060-8589, Japan. gett@anim.agr.hokudai.ac.jp
Watanabe
A
A
Tsunemitsu
H
H
Lee
N Y
NY
Kumura
H
H
Shimazaki
K I
KI
Yagi
Y
Y
D000818:Animals; D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents / Q000737:chemistry / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000276:immunology; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism; D007781:Lactoferrin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D018411:Spodoptera
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D006493:Heparin; D007781:Lactoferrin