title: Design, production, and characterization of a single-chain variable fragment (ScFv) derived from the prostate specific membrane antigen (PSMA) monoclonal antibody J591.
abstract: A single chain variable fragment (J591 ScFv) that recognizes the extracellular glyco-protein prostate specific membrane antigen (PSMA) was designed, constructed, and expressed in Pichia pastoris. Construction of the J591 ScFv was based on the reported complementarity-determining region (CDR) of the PSMA specific J591 monoclonal antibody (mAb). The nucleotide sequence encoding the J591-derived ScFv was codon-optimized for expression in P. pastoris and a 6× his-tag was added to facilitate affinity purification. A down-scale 2L methanol-induced P. pastoris fermentation yielded 330mg of total protein following a 96h induction. Following Immobolized Metal Affinity Chromatography, functionality of the J591 ScFv was confirmed via Western blot, immunoblot, binding studies, and flow cytometry analysis. The J591 ScFv showed binding affinity and specificity to cell extracts containing PSMA and PSMA-expressing prostate cancer cells. Our results demonstrate that functional J591 ScFv can be produced in P. pastoris for use in diagnostic and targeted therapeutic applications.
authors.affiliation: Graduate Field of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA. sp388@cornell.edu
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Parker
authors.forename: Ines Lopez-Calleja
authors.initials: IL
authors.lastname: Diaz
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Anderson
authors.forename: Carl A
authors.initials: CA
authors.lastname: Batt
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000954:Antigens, Surface / Q000276:immunology*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D043425:Glutamate Carboxypeptidase II / Q000276:immunology*; D006801:Humans; D008297:Male; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011467:Prostate / Q000166:cytology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C478680:J591 monoclonal antibody; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II