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 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 Mr on SDS-PAGE of 43,000 under nonreducing conditions. By contrast, the purified protein displayed a Mr 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 (Mr 43,000) and reducing (Mr 49,000) conditions.
Catimel
B
B
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Melbourne, Victoria 3050, Australia.
Ritter
G
G
Welt
S
S
Old
L J
LJ
Cohen
L
L
Nerrie
M A
MA
White
S J
SJ
Heath
J K
JK
Demediuk
B
B
Domagala
T
T
Lee
F T
FT
Scott
A M
AM
Tu
G F
GF
Ji
H
H
Moritz
R L
RL
Simpson
R J
RJ
Burgess
A W
AW
Nice
E C
EC
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003110:Colonic Neoplasms; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium; D005434:Flow Cytometry; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013501:Surface-Active Agents; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D010446:Peptide Fragments; D013501:Surface-Active Agents