Abstract
Objective: To characterize the expression of ST13 protein in human tissues for investigation of the function of colorectal cancer related gene ST13.Methods: ST13 ORF was cloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinity chromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinant protein. Immunoblot and immunohistochemical staining were employed to analyze ST13 protein expression in human tissues. Results:The expression and purification of the recombinant ST13 protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterial culture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from 104 to 105 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed that the apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility was approximately 50000, which was about 10000 larger than the 41324 calculated, but the glycosylation of the protein was excluded. Computer modeling revealed the protein to be a hydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributed in cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in the staining intensity of the protein were observed between normal and cancer tissues as well as among different normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with an apparent Mr of 50000. The protein is expressed in colorectal and other epithelial tissues. The expression level of the protein is down-regulated in colorectal cancer and varies among different normal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicates that ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibility that ST13 protein is involved in the development of colorectal cancer through Hsp70 molecular chaperone machinery.
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