Functional Studies Of The Protein Encoded By Esophageal Cancer Related Genel ECRG-1

A novel gene,esophageal cancer related gene l(ECRG-l), was isolated. The open reading frame of ECRG-1 is 1254bp,encoding a putative 418 amino acid protein. ECRG-1 is expressed in normal esophageal epithelium but not in the esophageal cancer.It can inhibit the proliferation of esophageal cancer cell.The findings indicate that ECRG-1 may play a role in the carcinogenesis and progression of human esophageal cancer.The work consists of four parts,the aim is to understand the function of ECRG-1 on many different levels. Part 1 was based on previous work,the fragmants of ECRG-1 were expressed in E.coli and purified.Two fragmants encode amino acid 40-418 and 175-418 of ECRG-1 protein were inserted into the pGEX-4T-l and PET-30a(+) vector.Fusion proteins were produced in BL21(DE3) and purified.The results demonstrated that GST-ECRG-1(175-418aa) was approximately 90% pure;the purity of GST-ECRG-1(40-418aa) was not satisfactory;the 6×His-ECRG-1(40-418aa) was expressed as predominantly as inclusion bodies in E.coli. Both GST-ECRG-1 (40-418aa) and GST-ECRG-1(175-418aa) did not display any serine protease activities.Part 2 identified the subcellular locolization of ECRG-1 protein.The green fluorescent protein was used as a flurorescent tag to determine the subcellular location of ECRG-1 protein. GFP was fused to either end of ECRG-1 protein using DNA recombinant technology, the constructs were transfected into Bel-...