Characterization Of The Protein Expressed By Colorectal Cancer Related Gene ST13/SNC6 In Human Tissues

Colorectal cancer (CRC) is one of the most common cancers andthe major causes of mortality in China. The normal mucosa-adenoma-carcinoma sequence has been accepted as the main pathway involvinga series of molecular changes, which include activation of oncogenes,inactivation of tumor suppressor genes and others in colorectalcarcinogenesis for several years. However, due to the complexity ofthe carcinogenesis, there are still many problems unsolved for themodel of colorectal carcinoma. All the known molecular events are notenough to explain fully the carcinogenesis of CRC. The identificationand characterization of novel human genes related to co1orectalcarcinoma might shed light on the mechanism of colorectal carcinomaand provide useful genetic markers for the early diagnosis andtre atm ent.In an attempt to seek out new genetic factors that are re1ated tothe development of CRC, Zheng' S reseach group has performedsubtractive hybridization between cDNA of normal mucosal tissuesand mRNA of colorectal carcinoma tissues from the same patient. Atotal of 46 cDNA clones whose expression was reduced in colorectalcancer as compared witl1 normal colorectal m ucosa were isolated andidentified. SNC6(Genbank Accession No.HSU l77l4), one of thesecDNA clones, has been further characterized on its structure andfunction, and nominated as STl3 (suppression of tumorigenicity l3)by HUGO Nomenclature Committee in 1998. The cDNA of STl3 geneis 3l45bp with an open reading frame encoding a protein of 369 aminoacids. STl3 gene has been mapped to chromosome 22ql3, and consistsof l2 exons and l l introns. The expression of this novel gene wasstudied by Dot b1ot' Northern blot \ RT-PCR-ELISA, in situhybridization and in vivo and in vitro transfection trial. Theexpression level of STl3 mRNA was lower in CRC as compared topaired normal intestinal mucosa, and varied in different cancer celllines and tissues. Growth restrain of CRC cell 1ines transfected bywild type STl3 gene was observed. Recently, STl3 gene has beenassigned to Human Genome Maps. AIl above indicate that STl3 genecan be further investigated as a novel colorectal negative related gene.However, protein is the final product of a gene and functions inall life activities. While the study of mRNA may show the expressionlevel and tissue distribution of a gene, there is inconsistency betweenthe expression of mRNA and its translated protein. The results ofmRNA cann't always explain the function of the gene. Therefore,further efforts have been made to reveal the structure and function ofthe protein encoded by STl3 gene in this study.l. Expression of STl3 gene in E.coli and purification of thefusion protein: To obtain enough amount of STl3 protein for furtherstudies, ST13 fu1l-Iength ORF, amp1ified by PCR, was cloned intoexpression vector pGEX-4T-2, and then expressed in E.coli. BamH lrestriction analysis and DNA sequencing showed that ST13ORF/pGEX-4T-2 recombinant plasmid was constructed successfullywith correct sequence. SDS-PAGE and anti-GST western blottingindicated that GST-STl3 protein was expressed in E.coli as expected.The fusion protein was purified by Glutathione Sepharose 4B affinitychromatography in a single step with a yield of 20% of total bacterialproteins, about 2.5mg GST-STl3 protein per liter of culture, and the-- - - -: - -. - - - ~'.Pul iLy lcu~hing 9l .3 %. Thrombin digestion was further done toremove the GST part from the fusion protein GST-STl3.2. Preparation of the monoclonal antibody against ST13 protein:To prepare the monoclonal antibody against STl3 protein, BALB/Cmice were immunized with the purified GST-STl3 protein. Three ce11strains of the monoclonal antibody were obtained by hybridomatechnique. Western blot confirmed that the antibodies specificallyrecognized ST13 protein in the transformed .E.coli lysates and theextracted total proteins of human tissues. The titers of the antib...