title: pPIC9-Fc: a vector system for the production of single-chain Fv-Fc fusions in Pichia pastoris as detection reagents in vitro.
abstract: Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates. For ScFv fragments, there is no such universal system available up to now. A vector system was constructed based on pPIC9- Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of mouse IgG1 and His-tag were cloned into the Pichia expression vector pPIC9. A model ScFv was introduced into pPIC9-Fc, which can bind Glutathione-S-transferase (GST) from Schistosoma japonicum, to yield the expression cassette pPIC9-ScFv-Fc. Following fermentation in a 5-liter reactor, the fusion was expressed at high levels in the methylotrophic yeast Pichia Pastoris, secreted as a dimeric form in the culture, and purified by Ni2+-NTA column chromatography. The expression yield can reach 10-30 mg/liter of culture medium. The ScFv-Fc fusion retains the biological binding ability of the parent ScFv, and can be applied as anti-GST antibodies for the detection of GST and GST-fusion proteins. Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-mouse antibodies.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Jianglan
authors.initials: J
authors.lastname: Liu
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
authors.forename: Feng
authors.initials: F
authors.lastname: Qian
authors.forename: Yuxun
authors.initials: Y
authors.lastname: Zhou
authors.forename: Jinzhi
authors.initials: J
authors.lastname: Wang
authors.forename: Yushu
authors.initials: Y
authors.lastname: Ma
authors.forename: Zeguang
authors.initials: Z
authors.lastname: Han
mesh_terms: D000818:Animals; D001666:Binding Sites, Antibody / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal / Q000276:immunology; D005822:Genetic Vectors; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D016254:Mutagenesis, Insertional; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins