Generation, purification, and characterization of a recombinant source of human prostate-specific antigen.
Human prostate-specific antigen (PSA), a 33- to 34-kDa serine proteinase with extensive homology to glandular kallikrein, is a single-chain glycoprotein that contains 7% carbohydrate. The presence of PSA in the serum of patients with prostatic cancer is widely employed as a marker of disease status. PSA has also been thought of as a possible target for use in active specific immunotherapy protocols. To date, the source of PSA employed has been seminal fluid from different individuals; this has raised concerns about differences among PSA batches for standardization of assays. This report is the first description of the production and the purification of a recombinant source of PSA using a baculovirus expression system. A baculovirus recombinant of the cDNA encoding the full length PSA was expressed in insect cells yielding two major immunoreactive products of 31 and 29 kDa. The latter size conforms to the molecular weight of a core preprotein deduced from the sequence of the cDNA insert. The larger protein represents the N-linked glycosylated form of the preprotein. Western blot analysis showed that both the glycosylated and aglycosylated forms of PSA reacted with a polyclonal and two different monoclonal antibodies specific for PSA. bV-PSA, like commercially available PSA, showed also low-molecular-weight immunoreactive products when culture supernatants were concentrated or taken through steps of purification. bV-PSA was purified to a final product consisting of a major 29-kDa protein and a minor 31-kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Bei
R
R
Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Paranavitana
C
C
Milenic
D
D
Kashmiri
S V
SV
Schlom
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D017430:Prostate-Specific Antigen / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D017430:Prostate-Specific Antigen