title: Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner.
abstract: Halophilic β-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649-658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The "Brevibacillus in vivo cloning" method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.
authors: Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan, tokunaga@chem.agri.kagoshima-u.ac.jp.
authors: Masao
authors: M
authors: Tokunaga
authors: Makoto
authors: M
authors: Mizukami
authors: Koji
authors: K
authors: Yamasaki
authors: Hiroko
authors: H
authors: Tokunaga
authors: Hiromasa
authors: H
authors: Onishi
authors: Hiroshi
authors: H
authors: Hanagata
authors: Matsujiro
authors: M
authors: Ishibashi
authors: Akira
authors: A
authors: Miyauchi
authors: Kouhei
authors: K
authors: Tsumoto
authors: Tsutomu
authors: T
authors: Arakawa
mesh_terms: D058675:Brevibacillus / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D001618:beta-Lactamases