Purification of human carcinoma antigen GA733-2 expressed in Escherichia coli and production of its polyclonal antibody in rabbit

Abstract

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.