The Construction And Expression Of Drosophila Selenoprotein Dselk Truncated Plasmid Expression In Human Cell And His-dselk Fusion Protein Expressed Plasmid Expression In Prokaryote

There are only three kinds of selenoproteins in Drosophila melanogaster.dSelK is one of them,which is a type III membrane protein.The N-terminus of dSelK is localized in the membrane and C-terminus is located in the Cytoplasm.And the human selenoprotein SelK is the homology of dSelK.The study of their biological functions just remains on the speculation.The preliminary study reveals that the dSelK can promote the release of Ca2+from endoplasmic reticulum to cytosol in Drosophila cells.The over-expression of dSelK can not only promote the release of Ca2+ from endoplasmic reticulum to cytosol in Drosophila cells,but also induce the apoptosis of human cancer cells.The similar function have verified for human selenoprotein SelK,the homology of dSelK,in human cancer cells.In the studies the human expression plasmids of truncated dSelK and SelK were constructed to confirm the special function of the protein sequence.The members of our lab had previously expressed the fusion protein s of dSelK and GST tag for the purification of dSelK protein,but the protein was abundant in inclusion body,which is hard for antibody preparation,purification,and structural study.So we changed tag of the fusion protein and tried to express the fusion protein His-dSelK in E coli.Drosophila selenoprotein dSelK and human selenoprotein SelK can be divided into three parts:the N-terminal sequence,the trans-membrane domain and C-terminal sequence.Because the selenocystine located C-terminal sequence generally is the functional sequence.Part of he C-terminal sequence of dSelK and SelK was truncated to construct the human expression plasmids and transfected into gastric cancer cells.The intracellular calcium changes and induction of the apoptosis of gastric cancer cells were observed.The pE-dSelK(Trun)-DsRed2 and pE-SelK(Trun)-DsRed plasmids were constructed and transfected into human gastric carcinoma 823 cells by lipoD293TM DNA In Virto Transfection Reagent and the fusion protein of dSelK(Trun)-DsRed and SelK(Trun)-DsRed were successfully expressed.Flow cytometry analysis revealed that the free Ca2+ in cytosol of the cancer cells which over expressed the fusion protein dSelK(Trun)-DsRed and SelK(Trun)-DsRed not had significant difference between the cancer cells over expressed only DsRed.On the other hand,the free Ca2+ in cytosol of the cancer cells which over expressed the fusion protein of the DsRed and the full length of dSelK and SelK was significantly increased.For the apoptosis studies of cancer cells,it was detected that the cancer cells which over expressed the fusion protein dSelK(Trun)-DsRed and SelK(Trun)-DsRed,as those cells over expressed only DsRed could not induce the apoptosis of cancer cells.On the other hand,the cancer cells which over expressed the fusion protein of the DsRed and the full length of dSelK and SelK could significantly induced the apoptosis of cancer cells.So the biological function of Drosophila dSelK and human SelK was verified.By inserting the mRNA sequence of dSelK into the pET-30a plasmid,the plasmid of pET-30a-dSelK was successfully constructed and over expressed in E coli.I got the fusion protein His-dSelK which was mainly the form of inclusion body,however a small amount of protein was still present in the supernatant.This provided a good condition for further separation and purification by His affinity column chromatography.Meanwhile due to the tag of His is much smaller than the tag of GST,it is comfortable for the production of a highly special antibody.