title: Highly efficient production of VHH antibody fragments in Brevibacillus choshinensis expression system.
abstract: Anti-IZUMO1PFF VHH (variable domain of camelid heavy chain antibody) clones, N6 and N15, from immunized alpaca (Lama pacos) phage library were efficiently expressed and their VHH products were secreted into the culture medium of Brevibacillus choshinensis HPD31-SP3, e.g., at a level of 26-95mg in 100ml conventional flask culture. With a 3-L scale fed-batch culture for 65h, the N15 VHH protein with C-terminal His-tag was produced at ∼3g/l culture medium. The N6 and N15 proteins were easily purified to apparent homogeneity by cation exchange and Ni-affinity chromatographies. Both proteins showed specific antigen-binding activity by ELISA and high antigen binding affinity, KD=6.0-8.6nM, by surface plasmon resonance analysis. Size exclusion chromatography-multi-angle laser light scattering analysis revealed that N6 and N15 proteins purified were exclusively monomeric form in phosphate buffered saline. CD spectrum showed beta-sheet rich structure, consistent with a typical antibody structure and also suggested aromatic-aromatic interactions, as indicated by a positive peak at 232nm. Thermal melting analysis of the N15 protein with C-terminal His-tag demonstrated a clear thermal transition with a Tm at 67°C. The heat-denatured sample recovered antigen binding activity upon cooling, indicating a reversible denaturation.
authors.affiliation: R & D Department, Higeta Shoyu Co., Ltd., 2-8 Chuo-cho, Choshi, Chiba 288-8680, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Mizukami
authors.affiliation: Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tokunaga
authors.affiliation: R & D Department, Higeta Shoyu Co., Ltd., 2-8 Chuo-cho, Choshi, Chiba 288-8680, Japan.
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Onishi
authors.affiliation: R & D Department, Higeta Shoyu Co., Ltd., 2-8 Chuo-cho, Choshi, Chiba 288-8680, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Ueno
authors.affiliation: R & D Department, Higeta Shoyu Co., Ltd., 2-8 Chuo-cho, Choshi, Chiba 288-8680, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hanagata
authors.affiliation: Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Miyazaki
authors.affiliation: Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Norihiko
authors.initials: N
authors.lastname: Kiyose
authors.affiliation: Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065, Japan. Electronic address: k2174603@kadai.jp.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Matsujiro
authors.initials: M
authors.lastname: Ishibashi
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Hagihara
authors.affiliation: Alliance Protein Laboratories, 6042 Cornerstone Court West, Suite A, San Diego, CA 92121, USA.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Arakawa
authors.affiliation: R & D Department, Higeta Shoyu Co., Ltd., 2-8 Chuo-cho, Choshi, Chiba 288-8680, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Miyauchi
authors.affiliation: Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. Electronic address: tokunaga@chem.agri.kagoshima-u.ac.jp.
authors.forename: Masao
authors.initials: M
authors.lastname: Tokunaga
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D058675:Brevibacillus / Q000235:genetics*; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies
keywords: Antibody fragments; Brevibacillus choshinensis; Heavy chain antibody; Reversible denaturation; Secretory production; VHH