The Structural And Functional Study Of Mutiple Transmembrane Protein-EMT-1

Seven-span transmembrane (7-TM) molecules constitute superfamilies of cell-surface proteins. They are receptors for small molecules .peptides ,hormones and ions .They also act as ligands for mediating a variety of external signals to the cytoplasm. For example, the signal transduction of G protein-coupled receptor is mediated by G protein and can generate intracellular messages;There exist some transmembrane proteins in the membrane of endoplasmic reticulum ,and some of them are related with the synthesis and transportation of some proteins, while others are related with substance metablism,such as glucose-6-phosphatase(G6Pase).In addition ,after binding with the 7-TM receptors affiliated to frizzled protein family,Wnt can activated disheveled protein in cytoplasm which is essential to embryonic development.In our studies,we have used the nuclear localization signalDepartment of Medical Genetics and Development Biology 4(NLS)selection system to isolate a novel gene from a mouse embryonic cDNA library. The full-length cDNA is about 1802bp and contains an open reading frame of 1296 nucleotides. It encodes 431 amino acid. The predicted secondary structure of the protein displays multiple putative transmembrane domains. The gene is (GenBank accession number is AF414190) named as ETM-l(endoplasmic reticulum-localized multi-span transmembrane protein-1) . The gene location in human chromosome is 6pll.2梡21.1 (GenBank accession number is All39392).The expression of the transcript of EMT-1 in various tissues was examined by Northern hybridization and MTE hybridization .The results showed that EMT-1 was expressed in multiple tissues, embryo and tumor cells. We fused the GFP into the C-terminal region as well as N-terminal region of EMT-1 protein .The result showed that the localization of the protein encoded by the full length EMT-1 cDNA was in endoplastic reticulum (ER) . Extracellular region , transmembrane and intracellular region showed the similar cellular localization .In order to get further evidence of the localization and distribution of EMT-1 in cells ,we have prepared EMT-1 His6-fusion protein and intend to abtain EMT-1 antibody for immuno- histochemistry assay. So, we subcloned EMT-1 gene into the prokaryotic fusion expression vector pRSETB and generated a 24ku protein .