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

  


Authors: Catimel, B.; Ritter, G.; Welt, S.; Old, L. J.; Cohen, L.; Nerrie, M. A.; White, S. J.; Heath, J. K.; Demediuk, B.; Domagala, T.; Lee, F. T.; Scott, A. M.; Tu, G. F.; Ji, H.; Moritz, R. L.; Simpson, R. J.; Burgess, A. W.; Nice, E. C.
Article Title: Purification and characterization of a novel restricted antigen expressed by normal and transformed human colonic epithelium
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.
Keywords: protein expression; human cell; flow cytometry; antigen expression; protein analysis; colonic neoplasms; tumor cells, cultured; blotting, western; membrane antigen; antibodies, monoclonal; amino acid sequence; molecular sequence data; cell transformation; peptide fragments; cell line, transformed; chromatography, high pressure liquid; epithelium; molecular weight; chromatography, gel; antigens, surface; polyacrylamide gel electrophoresis; electrophoresis, polyacrylamide gel; colon mucosa; chromatography, ion exchange; humans; human; priority journal; article; gel permeation chromatography; surface-active agents
Journal Title: Journal of Biological Chemistry
Volume: 271
Issue: 41
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 1996-10-11
Start Page: 25664
End Page: 25670
Language: English
DOI: 10.1074/jbc.271.41.25664
PUBMED: 8810343
PROVIDER: scopus
DOI/URL:

https://www.scopus.com/inward/record.uri?eid=2-s2.0-10144230010&doi=10.1074%2fjbc.271.41.25664&partnerID=40&md5=05d47682d45faa04df535b9c6b8783ae
Notes: Article -- Export Date: 22 November 2017 -- Source: Scopus
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MSK Authors
  1. Leonard Cohen
    18 Cohen
  2. Gerd Ritter
    166 Ritter
  3. Lloyd J Old
    593 Old
  4. Sydney Welt
    98 Welt
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