Construction, expression and characterization of a dual cancer-specific fusion protein targeting carcinoembryonic antigen in intestinal carcinomas.
The abundantly expressed carcinoembryonic antigen (CEA) on several cancer types is an attractive target for antibody-directed therapy. However, CEA is also present in some normal tissues. Here, we produced a dual functioning protein, designated as CAtin that exhibits both specific binding and killing functions, by fusing a tumor-specific apoptosis-inducing molecular Apoptin to C-terminus of an anti-CEA single-chain disulfide-stabilized Fv antibody (scdsFv). The CAtin proteins were expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni2+ affinity chromatography column. SDS-PAGE and Western blotting revealed that the recombinant protein was well-expressed and the yield was approximately 250mg/L. We demonstrated by flow cytometry and immunofluorescence assays that CAtin could bind specifically to human colon carcinoma cells (LoVo), but almost not to human uterine cervix (Hela). The results suggest that CAtin is active and specific toward CEA-positive cells and may potentially be used in CEA-targeted cancer therapy.
Yan
Liu
L
Department of Gastroenterology, Bethune Faculty of Medical Sciences of Jilin University, Changchun 130021, People's Republic of China.
Xiangwei
Meng
M
Xiao
Li
L
Peng
Gao
G
Chang
Li
L
Mingyao
Tian
T
Encheng
Yang
Y
Xiaohong
Xu
X
Peng
Jia
J
Shifu
Kan
K
Zhongmei
Wen
W
Ningyi
Jin
J
D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007414:Intestinal Neoplasms / Q000378:metabolism* / Q000628:therapy; D015514:Oncogene Proteins, Fusion / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary
D002272:Carcinoembryonic Antigen; D015514:Oncogene Proteins, Fusion