Studying On Cloning And Fusion Expression Of Enterokinase Light Chain Gene Used As Cutting Genetic Engineering Lable Protein

Enterokinase (EK) is a heterologous dimer serine protease esixsting in mammals' duodenal. The molecular weight is 150 kDa,and is made up of one 115 kDa heavy chain和one 35kD light chain. Natural intestinal kinase is made up of a structural subunit (chain) and a catalytic arc-(light chain) structure, both through an intermolecular disulfide combination of subunits will be responsible for the catalytic subunit fixed in the small intestine brush geo membrane and guide it to the intestinal movement, catalytic subunit can identify specific Asp-Asp-Asp-Asp-Lys sequence and the carboxy-terminal sequence along the cut, will be activated for trypsin trypsin, which initiates all zymogen activation of the cascade. and substrates specificity of protein hydrolysate with PH value fron 4. 5 to 9. 5 and temperature from 4 to 4 5℃.Since the EK cracking released by the fusion protein peptide with the purpose of wild-type and entirely consistent with N-terminal amino acid sequence, thus can be used as protein fusion protein expression system in the cutting reagent. But natural sources of intestinal kinase limited, and the high cost of extraction and separation, and the extraction of natural intestinal kinase easy for other protease pollution and cutting fusion protein degradation at the same time, the end products. And the method is genetic engineering can make up for these deficiencies, thus the use of genetic engineering production of intestinal kinase widely used. Recombinant intestinal light chain kinase (recombinant Enterokinase, rEKL) molecular mass of 35 kDa normally. REK proved to be all in vitro enzyme digestion and specificity compared to the natural intestinal kinase gene works on show at the end of the fusion protein enzyme activity increased, which is used for the production of genetically engineered intestinal kinase. In recent years more is done to Cloning and Expression of 235 amino acids light chainIn order to obtain the gene of wista rat enterokinase light chain ,which would be used in the cleavageand purification of fusion proteins. The fragment of wista rat enterokinase light chain cDNA was obtained by RT-PCR from wista rat' duodenal mucosa , then cloned into the pMD18-T cloning vector and sequenced. Compared with the sequence deposited in GenBank ,the cloned gene sequence is correct. Then the interested gene fragment was inserted into the pGEX-2T expression plasmid. The recombinant vector pGEX-rEKL was transformed into E. coli BL21 and induced by IPTG It was confirmed that the nucleotide sequence was correct on the conjunction site between the recombinant DNA 5'terminal multi2cloning site and recombinant fragment after the analysis of the nucleotide sequence. SDS-PAGE analysis indicated that target product was about 65 kDa which occupied 28 % of the total protein. A pure fusion protein was obtained by GST-sepharose chelating chromatogram. The successful cloning and expression in E. coli, a rat intestinal kinase gene, and the reorganization of the conditions of intestinal kinase expression optimizationThis investigation would be able to lay a foundation for enterokinase activity research and farther application of expression products on a large scale.