Production and purification of melanoma gp100 antigen and polyclonal antibodies.
gp100 is a melanoma-associated antigen found to carry immunogenic epitopes that can induce a CTL response against tumor cells. Production and purification of large quantities of this polypeptide may be important in the context of diagnosis and vaccinating against melanoma. To overcome the hydrophobic nature of gp100, we cloned and expressed only a part of the protein, and obtained a hydrophilic recombinant polypeptide (HR-gp100) that contained most of the immunogenic peptides. High yield was achieved in an Escherichia coli expression system. The protein was purified by AKTA Prime using anionic-columns. Polyclonal antibodies developed in chicken against HR-gp100 were efficient at detecting gp100 in melanoma cells, as determined by Western blot analysis and by immunohistochemistry. HR-gp100 can be used to develop a vaccine against melanoma. Antibodies to HR-gp100 may be used to detect tumors of melanocytic origin or to determine the level of gp100 expression in tumors prior to immunotherapy with the protein or one of its peptides.
MIGAL, South Industrial Area, P.O. Box 831, Kiryat Shmona 11016, Israel.
Y
Y
Gelbart
S
S
Frankenburg
Y
Y
Pinchasov
S
S
Krispel
D
D
Eliahu
O
O
Drize
E
E
Morag
D
D
Bartfeld
M
M
Lotem
T
T
Peretz
J
J
Pitcovski
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002645:Chickens / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007150:Immunohistochemistry; D008545:Melanoma / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology; D014407:Tumor Cells, Cultured; D058951:gp100 Melanoma Antigen
D000906:Antibodies; D000939:Epitopes; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D011994:Recombinant Proteins; D058951:gp100 Melanoma Antigen