title: Establishment of a mammalian expression system for recombinant [-2]proPSA and a specific antibody against the truncated leader peptide.
abstract: A truncated precursor form of prostate-specific antigen (PSA), [-2]proPSA, is a well-known biomarker for prostate cancer. To develop a biomarker assay, highly purified [-2]proPSA is required as a standard reference and for generation of a specific antibody. In this study, we generated an efficient mammalian expression system for producing a recombinant [-2]proPSA-human kappa constant domain (Cκ) fusion protein. N-terminal amino acid sequencing using Edman degradation demonstrated that over 95% of the recombinant protein produced is [-2]proPSA, thereby showing for the first time that recombinant [-2]proPSA can be produced as a major fraction. We also generated a recombinant chicken antibody specific to [-2]proPSA but not cross-reactive to recombinant [-7]proPSA-Cκ, [-5]proPSA-Cκ, and PSA purified from human seminal fluid in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Also, the recombinant chicken antibody reacted to recombinant [-2]proPSA protein bound to an anti-PSA antibody coated on the micrometer plate in a sandwich ELISA. All of these results suggest that the N-terminus of the [-2]proPSA-Cκfusion protein resides on the exterior of the protein, thus allowing exposure to the antibody.
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Dobeen
authors.initials: D
authors.lastname: Hwang
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Aerin
authors.initials: A
authors.lastname: Yoon
authors.affiliation: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Soohyun
authors.initials: S
authors.lastname: Kim
authors.affiliation: Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Hyori
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.|Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Junho
authors.initials: J
authors.lastname: Chung
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D030161:Avian Proteins / Q000737:chemistry* / Q000276:immunology; D002645:Chickens; D057809:HEK293 Cells; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007610:Kallikreins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D017430:Prostate-Specific Antigen / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D030161:Avian Proteins; D007145:Immunoglobulin kappa-Chains; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen
keywords: [-2]proPSA; antibody; prostate cancer; recombinant expression system