Purification and characterization of a novel restricted antigen expressed by normal and transformed human colonic epithelium
- Catimel, B; Ritter, G; Welt, S; Old, LJ; Cohen, L; Nerrie, MA; White, SJ; Heath, JK; Demediuk, B; Domagala, T; Lee, FT; Scott, AM; Tu, GF; Ji, H; Moritz, RL; Simpson, RJ; Burgess, AW; Nice, EC;
Publication Year 1996-10-11, Volume 271, Issue #41, Page 25664-25670
- Journal Title
- JOURNAL OF BIOLOGICAL CHEMISTRY
- Publication Type
- Journal Article
- 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 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 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: ISVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD. 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.
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
- SURFACE-PLASMON RESONANCE; QUANTITATIVE-ANALYSIS; PROTEIN; CANCER; BIOSENSOR; A33
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Creation Date: 1996-10-11 12:00:00Last Modified: 0001-01-01 12:00:00