Detection of a putative 30-kDa ligand of the cluster-2 antigen.
The cluster-2 antigen, also called EGP-2, is a 38-kDa transmembrane glycoprotein with a distribution that is largely confined to human epithelial cells and their derived carcinomas. Monoclonal antibodies (MAbs) directed against EGP-2 have been extensively studied as anti-tumor agents, yet the function of the antigen is not known. In the present study we used a biotinylated recombinant soluble derivative of the EGP-2 (sEGPbio) as a probe to detect a possible EGP-2 ligand, using various carcinoma cell lines as a substrate. The recombinant soluble EGP-2 was expressed in the Autographa californica nuclear polyhedrosis virus (baculovirus) expression system. The sEGP-2, to which we engineered a poly-histidine affinity tag, was purified from infected Spodoptera frugiperda insect cells using immobilized metal-ion-affinity chromatography (IMAC). In Western blot analysis the sEGPbio probe bound to a 30-kDa protein band in 2 out of 5 of the assessed carcinoma cell lines, suggesting that this band may be an EGP-2 ligand. Interestingly, binding only occurred when, prior to SDS-PAGE, cell lysates had been subjected to a reducing agent (2-mercapto-ethanol). The physiological significance of this phenomenon and nature of the detected 30-kDa protein band remains to be determined.
Helfrich
W
W
Department of Clinical Immunology, University Hospital Groningen, The Netherlands.
Van Geel
M
M
The
T H
TH
De Leij
L
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D018288:Carcinoma, Small Cell; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D000071858:Epithelial Cell Adhesion Molecule; D005822:Genetic Vectors; D006801:Humans; D007124:Immunoenzyme Techniques; D008024:Ligands; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D016254:Mutagenesis, Insertional; D017924:Nucleopolyhedroviruses; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins