PURIFICATION AND CHARACTERIZATION OF A NOVEL RESTRICTED ANTIGEN EXPRESSED BY NORMAL AND TRANSFORMED HUMAN COLONIC EPITHELIUM

A cell surface antigen that is expressed by normal and 95% of transfor med colonic epithelium and is recognized by the monoclonal antibody A3 3 (Welt, S., Divgi, C. R., Real F. X., Yeh, S. D., Garin-Chesa, P., Fi nstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., C arswell, E. A., Oettgen, H. F., and Old, L. J. (1990) J. Clin. Oncol. 8, 1894-1906) has been purified to homogeneity from the human colonic carcinoma cell line LIM1215. The A33 protein was purified fi om Triton X-114 extracts of LIM1215 cells under nondenaturing conditions. These extracts were applied sequentially to Green-Sepharose HE-4BD, Mono-Q HR 10/10, Superose 12 HR 10/30, and micropreparative Brownlee Aquapore RP 300. The purification was monitored by biosensor analysis using su rface plasmon resonance detection with a F(ab')(2) fragment of the hum anized A33 monoclonal antibody immobilized on the sensor surface and W estern blot analysis following SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions using humanized A33 monoclonal ant ibody. The purified A33 antigen has a M(r) on SDS-PAGE of 43,000 under nonreducing conditions. By contrast, the purified protein displayed a M(r) of approximately 180,000 under native conditions on both size ex clusion chromatography and native PAGE, possibly due to the formation of a homotetramer. N-terminal amino acid sequence analysis of the puri fied protein identified 34 amino acid residues of a unique sequence: I SVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD. A polyclonal antibody was raise d against a synthetic peptide corresponding to residues 2-20 of this s equence. The antipeptide serum recognized the purified protein using W estern blot analysis under both nonreducing (M(r) 43,000) and reducing (M(r) 49,000) conditions.