TY - JOUR
T1 - Purification and characterization of a novel restricted antigen expressed by normal and transformed human colonic epithelium
AU - Catimel, B.
AU - Ritter, G.
AU - Welt, S.
AU - Old, L. J.
AU - Cohen, L.
AU - Nerrie, M. A.
AU - White, S. J.
AU - Heath, J. K.
AU - Demediuk, B.
AU - Domagala, T.
AU - Lee, F. T.
AU - Scott, A. M.
AU - Tu, G. F.
AU - Ji, H.
AU - Moritz, R. L.
AU - Simpson, R. J.
AU - Burgess, A. W.
AU - Nice, E. C.
PY - 1996/10/19
Y1 - 1996/10/19
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=10144230010&partnerID=8YFLogxK
U2 - 10.1074/jbc.271.41.25664
DO - 10.1074/jbc.271.41.25664
M3 - Article
C2 - 8810343
AN - SCOPUS:10144230010
SN - 0021-9258
VL - 271
SP - 25664
EP - 25670
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 41
ER -