title: Single-chain variable fragment antibody against human aspartyl/asparaginyl beta-hydroxylase expressed in recombinant Escherichia coli.
abstract: The human aspartyl beta-hydroxylase (HAAH) is a highly conserved enzyme that hydroxylates epidermal growth factor-like domains in transformation-associated proteins. Previous studies showed that the gene of HAAH was overexpressed in many human malignancies. In the present study, the HAAH-specific single-chain variable fragment (ScFv) antibody was produced in recombinant Escherichia coli. The variable regions of the genes of the heavy chain (VH) and light chain (VL) cloned from the hybridoma cells G3/F11 were connected with a flexible linker using an overlap extension polymerase chain reaction. Nucleotide sequence analysis revealed that the anti-HAAH VH was a member of the VH V gene family and the VL gene belonged to the Vκ gene family VI subgroup. Extensive efforts to express the functional ScFv antibody in E. coli have been made by using two different prokaryotic expression vectors-pHEN1 and pET-16b-to compare the expression level and solubility of the antibody. The recombinant pHEN1/E1-anti-HAAH vector could express soluble ScFv, although the yield was only 7.8% of the total cellular protein. However, the pET-16b/E2-anti-HAAH vector produced the ScFv as inclusion bodies inside the host cytoplasm, although the expression level of the antibody was quite high (28.5% of the total cellular protein). Soluble ScFv antibody produced by pHEN1/E1-anti-HAAH was characterized for its antigen-binding characteristics. Its antigen affinity as antibody was measured by indirect enzyme linked immunosorbent assay analysis and proved to have high binding activity to the antigen HAAH.
authors.affiliation: Faculty of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi'an, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.forename: Hua
authors.initials: H
authors.lastname: Wang
authors.forename: Tian
authors.initials: T
authors.lastname: Xue
authors.forename: Xiao-ping
authors.initials: XP
authors.lastname: Xue
authors.forename: Ting
authors.initials: T
authors.lastname: Huyan
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Kai
authors.initials: K
authors.lastname: Song
mesh_terms: D000915:Antibody Affinity / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006899:Mixed Function Oxygenases / Q000276:immunology*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D057127:Single-Chain Antibodies; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase