Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner

• Published in: Applied microbiology and biotechnology Vol. 97; no. 19; pp. 8569 - 8580
• Main Authors: Tokunaga, Masao, Mizukami, Makoto, Yamasaki, Koji, Tokunaga, Hiroko, Onishi, Hiromasa, Hanagata, Hiroshi, Ishibashi, Matsujiro, Miyauchi, Akira, Tsumoto, Kouhei, Arakawa, Tsutomu
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.10.2013; Springer; Springer Nature B.V
• Subjects: Amino acids; Antibodies; Antigens; beta-Lactamases - genetics; beta-Lactamases - metabolism; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Brevibacillus; Brevibacillus - genetics; Brevibacillus - metabolism; Chains; Cloning; E coli; Enzymatic activity; Escherichia coli; Fluorescein; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - isolation & purification; Immunoglobulin Variable Region - metabolism; Laboratories; Life Sciences; Light scattering; Microbial Genetics and Genomics; Microbiology; Microorganisms; Molecular structure; Molecular Weight; Monoclonal antibodies; Plasmids; Protein synthesis; Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; Single-Chain Antibodies - isolation & purification; Single-Chain Antibodies - metabolism; Studies; Thrombin; Viral antibodies; Japan; Net negative charge; High solubility; Halophilic β-lactamase; Single-chain antibody (scFv); Fusion protein
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-013-4695-2

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.