Generation of a baculovirus recombinant prostate-specific membrane antigen and its use in the development of a novel protein biochip quantitative immunoassay.
Prostate-specific membrane antigen (PSMA) is a 100-kDa transmembrane glycoprotein identified by the monoclonal antibody 7E11-C5.3 from the human prostate tumor cell line LNCaP. Because of its significant upregulation in androgen refractory and metastatic prostate cancers, PSMA may be a useful prognostic biomarker and a target for developing novel therapeutic strategies. However, the lack of abundant pure PSMA protein and the low efficacy in immunoaffinity isolation from LNCaP cells have hampered the development of clinical assays. In order to obtain a renewable and reliable source of pure antigen, we used the baculovirus/insect cell system to express and purify a recombinant PSMA. A recombinant baculovirus containing a 6x histidine-tagged PSMA gene was generated, from which rPSMA was expressed and purified using cobalt-chelating affinity chromatography. The purity and correct molecular size of rPSMA were demonstrated by gel electrophoresis and mass spectrometry. Glycosidase digestions showed that the oligosaccharides on rPSMA are primarily N-linked high-mannose type. Although the glycosylation is different from the native PSMA, it did not affect the immunoreactivity of rPSMA to antibodies specific for either the intra- or the extracellular domains of PSMA. Finally, the purified rPSMA was successfully used to develop a quantitative PSMA immunoassay using the novel ProteinChip surface-enhanced laser desorption/ionization mass spectrometry technology.
Xiao
Z
Z
Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.
Jiang
X
X
Beckett
M L
ML
Wright
G L
GL
D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D000954:Antigens, Surface; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002268:Carboxypeptidases / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D005822:Genetic Vectors; D043425:Glutamate Carboxypeptidase II; D006031:Glycosylation; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007915:Lepidoptera / Q000166:cytology; D011993:Recombinant Fusion Proteins / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D011993:Recombinant Fusion Proteins; D002268:Carboxypeptidases; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II