title: Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants.
abstract: Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.
authors.affiliation: Medizinische Klinik III, Hematology, Oncology und Transfusion Medicine, Charité, Campus Benjamin Franklin, Hindenburgdamm 30, 12200, Berlin, Germany.
authors.forename: Hossein
authors.initials: H
authors.lastname: Panjideh
authors.forename: Vânia
authors.initials: V
authors.lastname: Coelho
authors.forename: Jens
authors.initials: J
authors.lastname: Dernedde
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fuchs
authors.forename: Ulrich
authors.initials: U
authors.lastname: Keilholz
authors.forename: Eckhard
authors.initials: E
authors.lastname: Thiel
authors.forename: P Markus
authors.initials: PM
authors.lastname: Deckert
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D018533:Biomass; D019149:Bioreactors; D043525:Cytosine Deaminase / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004798:Enzymes / Q000737:chemistry; D005285:Fermentation; D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000906:Antibodies; D004798:Enzymes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D043525:Cytosine Deaminase; D010100:Oxygen; D000432:Methanol