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KMID : 1161520150190030188
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Animal Cells and Systems
2015 Volume.19 No. 3 p.188 ~ p.193
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Purification of human carcinoma antigen GA733-2 expressed in Escherichia coli and production of its polyclonal antibody in rabbit
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Park Se-Hee
Kim Ah-Young
Ma Sang-Hoon
Kim Hyun-Min
Kang Hyung-Sik
Maeng Jin-Soo
Ko Ki-Sung
Chung In-Sik
Joung Young-Hee
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Abstract
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GA733-2, an epithelial cell adhesion molecule highly expressed at the human colorectal carcinoma cell surface, is a candidate protein for the development of a colorectal cancer preventive vaccine. In this study, a signal peptide and c-terminal transmembrane domain truncated GA733-2 was fused to a His-tag, and the recombinant protein (rGA733-2) was expressed in Escherichia coli Rosetta (DE3) pLysS. rGA733-2 was expressed in the form of inclusion bodies, and the protein was purified using either a serial dialyzed urea refolding method or metal ion exchange column after 8 M urea solubilization. The efficiency of rGA733-2 purification was increased three times using the urea refolding method compared to the column purification. A polyclonal antibody against the rGA733-2 was generated in rabbit using the refolded rGA733-2 as an antigen. The polyclonal antibodies (pAb rGA733-2) sensitively detected rGA733-2, which was heterologously expressed in bacteria and plants, and also detected native GA733-2 proteins in mouse and human cancer cells. pAb rGA733-2 showed high specificity against tobacco-expressed rGA733-2 in an immunoprecipitation assay. rGA733-2 and pAb rGA733-2 could serve as useful tools toward the development of a plant-derived colorectal cancer vaccine.
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KEYWORD
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carcinoma antigen GA733-2, polyclonal antibody against rGA733-2, urea refolding, tobacco-expressed rGA733-2
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