Production of an anti-prostate-specific antigen single-chain antibody fragment from Pichia pastoris.
Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. Because PSA levels are normally quite low, an antibody-based assay must be used to detect PSA. However, not all PSA-specific antibodies bind equally well to PSA or to its different isoforms. Therefore, a better understanding of how PSA interacts with PSA-specific antibodies is of considerable clinical interest. B80.3 is a widely used murine monoclonal anti-PSA antibody (IgG), which has very high affinity for both free and alpha-anti-chymotrypsin complexed PSA. More importantly, its gene sequence is known-making it one of only two anti-PSA antibodies that has been fully cloned and sequenced. To better elucidate the interaction between PSA and B80.3, a single-chain antibody fragment, derived from the variable domain of B80.3 (scFvB80), was cloned into a pPIC9 vector and expressed in Pichia pastoris. The secreted protein was purified using a three-step protocol beginning with a 50% ammonium sulfate precipitation step, followed by a T-gel thio-affinity step and concluding with a simple anion-exchange (DE52) filtration step. NMR studies indicate the protein is correctly folded while competitive enzyme-linked immunosorbant assays show that the purified scFvB80 has approximately 20% of the activity of the full-length B80.3 antibody. The protocol described here provides a quick and convenient route to prepare large quantities of very pure anti-PSA antibody fragments (15-20 mg/L culture medium) for detailed structural and biophysical characterization.
Wang
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Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada.
Wang
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Jette
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Wishart
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics*; D017430:Prostate-Specific Antigen / Q000276:immunology*; D017510:Protein Folding; D014170:Transformation, Genetic
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D017430:Prostate-Specific Antigen