title: Screening of single-chain variable fragments against TSP50 from a phage display antibody library and their expression as soluble proteins.
abstract: A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein inEscherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.
authors.affiliation: College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun, 130021, China. jingyanwei@yahoo.com.cn
authors.forename: Jingyan
authors.initials: J
authors.lastname: Wei
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.forename: Songchuan
authors.initials: S
authors.lastname: Yang
authors.forename: Junjie
authors.initials: J
authors.lastname: Xu
authors.forename: Hangtian
authors.initials: H
authors.lastname: Kong
authors.forename: Bing
authors.initials: B
authors.lastname: Han
authors.forename: Yongli
authors.initials: Y
authors.lastname: Bao
authors.forename: Yin
authors.initials: Y
authors.lastname: Wu
authors.forename: Weitian
authors.initials: W
authors.lastname: Yin
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Ganglin
authors.initials: G
authors.lastname: Yan
authors.forename: Guimin
authors.initials: G
authors.lastname: Luo
authors.forename: Hao-Peng
authors.initials: HP
authors.lastname: Xu
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Li
authors.forename: Bai
authors.initials: B
authors.lastname: Yang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D001943:Breast Neoplasms / Q000175:diagnosis / Q000276:immunology; D003090:Coliphages / Q000276:immunology; D004926:Escherichia coli; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D051379:Mice; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D012697:Serine Endopeptidases / Q000276:immunology* / Q000378:metabolism*; D012995:Solubility
chemical_list: D000906:Antibodies; D015415:Biomarkers; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C446379:testis-specific protease 50