title: Construction, expression and characterization of a dual cancer-specific fusion protein targeting carcinoembryonic antigen in intestinal carcinomas.
abstract: 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.
authors.affiliation: Department of Gastroenterology, Bethune Faculty of Medical Sciences of Jilin University, Changchun 130021, People's Republic of China.
authors.forename: Liu
authors.initials: L
authors.lastname: Yan
authors.forename: Meng
authors.initials: M
authors.lastname: Xiangwei
authors.forename: Li
authors.initials: L
authors.lastname: Xiao
authors.forename: Gao
authors.initials: G
authors.lastname: Peng
authors.forename: Li
authors.initials: L
authors.lastname: Chang
authors.forename: Tian
authors.initials: T
authors.lastname: Mingyao
authors.forename: Yang
authors.initials: Y
authors.lastname: Encheng
authors.forename: Xu
authors.initials: X
authors.lastname: Xiaohong
authors.forename: Jia
authors.initials: J
authors.lastname: Peng
authors.forename: Kan
authors.initials: K
authors.lastname: Shifu
authors.forename: Wen
authors.initials: W
authors.lastname: Zhongmei
authors.forename: Jin
authors.initials: J
authors.lastname: Ningyi
mesh_terms: 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
chemical_list: D002272:Carcinoembryonic Antigen; D015514:Oncogene Proteins, Fusion