Purification and characterization of a novel restricted antigen expressed by normal and transformed human colonic epithelium

B. Catimel, G. Ritter, S. Welt, L. J. Old, L. Cohen, M. A. Nerrie, S. J. White, J. K. Heath, B. Demediuk, T. Domagala, F. T. Lee, A. M. Scott, G. F. Tu, H. Ji, R. L. Moritz, R. J. Simpson, A. W. Burgess, E. C. Nice

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Abstract

A cell surface antigen that is expressed by normal and 95% of transformed colonic epithelium and is recognized by the monoclonal antibody A33 (Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Garin-Chesa, P., Finstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, 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 from 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 surface plasmon resonance detection with a F(ab')2 fragment of the humanized A33 monoclonal antibody immobilized on the sensor surface and Western blot analysis following SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions using humanized A33 monoclonal antibody. 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 exclusion chromatography and native PAGE, possibly due to the formation of a homotetramer. N-terminal amino acid sequence analysis of the purified protein identified 34 amino acid residues of a unique sequence: ISVETPQDVLRASQGKS-VTLPXTYHTSXXXREGLIQWD. A polyclonal antibody was raised against a synthetic peptide corresponding to residues 2-20 of this sequence. The antipeptide serum recognized the purified protein using Western blot analysis under both nonreducing (M(r) 43,000) and reducing (M(r) 49,000) conditions.

Original languageEnglish
Pages (from-to)25664-25670
Number of pages7
JournalThe Journal of Biological Chemistry
Volume271
Issue number41
DOIs
Publication statusPublished - 19 Oct 1996
Externally publishedYes

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