title: Expression, purification and characterization of a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen in Pichia pastoris.
abstract: Because the demand for rabies post exposure prophylaxis (PEP) treatment has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide an adequate amount of the required passive immune component in PEP in countries where canine rabies is endemic. The replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for the treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen to develop a product that can be used as a component of the PEP cocktail. We cloned the ScFv fragment from a human ScFv library that was established previously and inserted this fragment into the expression vector pPICZαC/Fc. An active recombinant ScFv-Fc fusion protein was successfully expressed in Pichia pastoris. The production of ScFv-Fc was optimized and scaled up in an 80L fermentor with yields exceeding 60mg/L. The ScFv-Fc protein was purified to more than 95% purity using a two-step scheme: ammonium sulfate fractionation and Protein A Sepharose CL-4B. The ScFv-Fc fusion protein neutralized rabies virus in a standard in vivo neutralization assay in which the virus was incubated with the ScFv-Fc molecules before intracranial inoculation in mice. Our results suggest that functional antibodies can be produced in P. pastoris and that ScFv-Fc fusion proteins have the potential to serve as therapeutic candidates.
authors.affiliation: Department of Biotechnology, Institute of Life Science and Biopharmacology, Guangdong Pharmaceutical University, Guangzhou 510006, China. wangdingding305@yahoo.com.cn
authors.forename: Ding-ding
authors.initials: DD
authors.lastname: Wang
authors.forename: Man-man
authors.initials: MM
authors.lastname: Su
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.forename: Shu-lin
authors.initials: SL
authors.lastname: Huang
authors.forename: Ju
authors.initials: J
authors.lastname: Wang
authors.forename: Wei-qun
authors.initials: WQ
authors.lastname: Yan
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D007928:Lethal Dose 50; D051379:Mice; D009500:Neutralization Tests; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011820:Rabies virus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies