title: High-level secretory production of recombinant single-chain variable fragment (scFv) in Corynebacterium glutamicum.
abstract: We describe the development of a new secretory production system for the enhanced production of a single-chain variable fragment (scFv) against the anthrax toxin in Corynebacterium glutamicum. For efficient secretory production of the antibody fragment, the following components were examined: (1) signal peptides, (2) codon usage of antibody fragment, (3) promoters, (4) 5' untranslated region (5' UTR) sequence, and (5) transcriptional terminator. Among all the systems examined, the use of a codon-optimized gene sequence, a Sec-dependent PorB signal peptide, and a fully synthetic H36 promoter, allowed the highest production of antibody fragments in a culture medium. For large-scale production, fed-batch cultivations were also conducted in a 5-L lab-scale bioreactor. When cells were cultivated in semi-defined media, cells could grow up to an OD600 of 179 for 32 h and an antibody fragment concentration as high as 68 mg/L could be obtained in a culture medium with high purity. From the culture medium, the secreted antibody was successfully purified using a simple purification procedure, with correct binding activity confirmed by enzyme-linked immunosorbent assay. To the best of our knowledge, this is the first report of a fed-batch cultivation for antibody fragment production in C. glutamicum.
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea.
authors.forename: Sung Sun
authors.initials: SS
authors.lastname: Yim
authors.forename: Seul Ji
authors.initials: SJ
authors.lastname: An
authors.forename: Jae Woong
authors.initials: JW
authors.lastname: Choi
authors.forename: Ae Jin
authors.initials: AJ
authors.lastname: Ryu
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
mesh_terms: D000907:Antibodies, Bacterial / Q000235:genetics / Q000378:metabolism; D000942:Antigens, Bacterial / Q000276:immunology; D001427:Bacterial Toxins / Q000276:immunology; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C030325:anthrax toxin