title: Improving the productivity of single-chain Fv antibody against c-Met by rearranging the order of its variable domains.
abstract: Single-chain Fv (scFv) antibody against c-Met is expected to be employed in clinical treatment or imaging of cancer cells owing to the important biological roles of c-Met in the proliferation of malignancies. Here, we show that the productivity of scFv against c-Met in Escherichia coli is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders (i.e., VL-linker-VH and VH-linker-VL), expressed them in the cytoplasm of E. coli trx/ gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with VH/VL orientation was more than five times higher than that with VL/VH format. Coexpression of DsbC enhanced the yield of soluble amounts of anti-c-Met scFv protein for both constructs. The purified scFv antibodies of the two different formats exhibited almost the same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with VH/VL or VL/VH formats and obtained similar results to the case of scFv antibodies.
authors.affiliation: Department of Polymer Science and Chemical Engineering, Pusan National University, Busan, Korea.
authors.forename: Yu-Jin
authors.initials: YJ
authors.lastname: Kim
authors.forename: Rameshkumar
authors.initials: R
authors.lastname: Neelamegam
authors.forename: Mi-Ae
authors.initials: MA
authors.lastname: Heo
authors.forename: Selvakumar
authors.initials: S
authors.lastname: Edwardraja
authors.forename: Hyun-Jong
authors.initials: HJ
authors.lastname: Paik
authors.forename: Sun-Gu
authors.initials: SG
authors.lastname: Lee
mesh_terms: D018033:Antibodies, Bispecific / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000276:immunology; D000915:Antibody Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D019859:Proto-Oncogene Proteins c-met / Q000276:immunology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D018033:Antibodies, Bispecific; D000912:Antibodies, Neoplasm; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv; D019859:Proto-Oncogene Proteins c-met