Protein Expression and Purification 21, 424–431 (2001) doi:10.1006/prep.2001.1396, available online at http://www.idealibrary.com on Production of Recombinant Bovine Lactoferrin N-Lobe in Insect Cells and Its Antimicrobial Activity Ichiro Nakamura,* , † ,1 Atsushi Watanabe,† Hiroshi Tsunemitsu,‡ Nai-Yuan Lee,* Haruto Kumura,* Kei-ichi Shimazaki,* and Yukio Yagi† *Dairy Science Laboratory, Faculty of Agriculture, Hokkaido University, N9W9 Kita-Ku, Sapporo, Hokkaido 060-8589, Japan; †Laboratory of Clinical Biochemistry, Hokkaido Research Station, National Institute of Animal Health, Hitsujiga-oka Toyohira-Ku, Sapporo, Hokkaido 062-0045, Japan; and ‡Shichinohe Research Unit, National Institute of Animal Health, Shichinohe-Cho, Kamikita-Gun, Aomori 039-2586, Japan Received September 29, 2000, and in revised form December 12, 2000 N-lobe (the N-terminal half) and the C-lobe (the C- Lactoferrin is a multifunctional, iron-binding glyco- terminal half). The two lobes are connected by a short protein found in physiological fluids of mammals. In “hinge” peptide and each lobe has one iron-binding site the present study, a gene encoding the N-terminal half (3, 4). (N-lobe) of bovine lactoferrin was cloned and expressed Lactoferrin has been attributed many physiological in cultured insect cells using a baculovirus expression roles such as serving as a regulator of iron metabolism, system. One mutation was found in the lactoferrin N- a component of the host defense system against infec- lobe gene, but it resulted in no amino acid substitution. tions, a nonspecific mediator of inflammation, and so The recombinant lactoferrin N-lobe was secreted into on. These are related to various functional properties the culture medium and partially purified by means of lactoferrin such as its iron-binding activity; its bacte- of an immobilized heparin column. The recombinant riostatic (or bactericidal) activity; its cell-binding activ- lactoferrin N-lobe secreted was not glycosylated, but ity (binding to cell surface receptors); its ability to bind it possessed antimicrobial activity toward Escherichia to various biological materials such as ferritin (5), DNA coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electro- (6), heparin (7), and lipopolysaccharide (8, 9); its activa- phoretic mobility on SDS–PAGE than the secreted tion of some kinds of leukocytes; and so on (10). Many product and showed no potency to inhibit the growth of these functions of lactoferrin are thought to be in- of bacteria. It is thought that the product accumulated volved in the N-lobe (11, 12). The functions of lactoferrin intracellularly lacks antimicrobial ability due to its and its biological roles must be further elucidated for degradation in the host cells or due to disruption of the commercial application of this protein. the active conformation.  2001 Academic Press Separation of the N-lobe from the C-lobe is a useful Key Words: bovine lactoferrin; antimicrobial activity; approach to study the structure–function relationship baculovirus; insect cells; milk protein. of lactoferrin. However, it has been found to be very difficult to separate the two lobes by digestion with proteolytic enzymes or by chemical cleavage because Lactoferrin is an iron-binding glycoprotein found in one of the peptide bonds (K282–S283) in the N-lobe is physiological fluids (1) such as milk and in the specific very susceptible to digestion by trypsin (13). In the granules of neutrophils (2). Lactoferrin consists of a present study, cloning and expression of a lactoferrin N- single polypeptide chain (approximately 80 kDa) folded lobe (LfN) 2 gene were performed and the antimicrobial into two structurally homologous lobes designated the activity of the recombinant LfN was examined. 1 To whom correspondence and reprint requests should be ad- dressed at Dairy Science Laboratory, Hokkaido University, N9W9 2 Abbreviations used: LfN, lactoferrin N-lobe; PCR, polymerase chain reaction; AcNPV, Autographa californica nuclear polyhedrosis Kita-Ku, Sapporo, Hokkaido 060-8589, Japan. Fax: +81-11-706-4135. E-mail: gett@anim.agr.hokudai.ac.jp. virus; Sf-9, Spodoptera frugiperda 9; HRP, horseradish peroxidase; 424 1046-5928/01 $35.00 Copyright  2001 by Academic Press All rights of reproduction in any form reserved.