Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombi...Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinitychromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinantprotein. Immunoblot and immunohistochemical staining were employed to analyze ST13 proteinexpression in human tissues. Results: The expression and purification of the recombinant ST13protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterialculture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from10~4 to 10~5 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed thatthe apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility wasapproximately 50 000, which was about 10 000 larger than the 41 324 calculated, but theglycosylation of the protein was excluded. Computer modeling revealed the protein to be ahydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributedin cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in thestaining intensity of the protein were observed between normal and cancer tissues as well as amongdifferent normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with anapparent Mr of 50 000. The protein is expressed in colorectal and other epithelial tissues. Theexpression level of the protein is down-regulated in colorectal cancer and varies among differentnormal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicatesthat ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibilitythat ST13 protein is involved in the development of colorectal cancer through Hsp70 molecularchaperone machinery.展开更多
文摘Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinitychromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinantprotein. Immunoblot and immunohistochemical staining were employed to analyze ST13 proteinexpression in human tissues. Results: The expression and purification of the recombinant ST13protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterialculture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from10~4 to 10~5 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed thatthe apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility wasapproximately 50 000, which was about 10 000 larger than the 41 324 calculated, but theglycosylation of the protein was excluded. Computer modeling revealed the protein to be ahydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributedin cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in thestaining intensity of the protein were observed between normal and cancer tissues as well as amongdifferent normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with anapparent Mr of 50 000. The protein is expressed in colorectal and other epithelial tissues. Theexpression level of the protein is down-regulated in colorectal cancer and varies among differentnormal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicatesthat ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibilitythat ST13 protein is involved in the development of colorectal cancer through Hsp70 molecularchaperone machinery.
