Prokaryotic And Pichia Pastoris Expression Of EPA Gene

Cerebrovascular disease is one of the principal diseases harmful to human life and health which shows driving rising tendency. It is very important and significant to find new medicine to prevent cerebrovascular diseases. Lumbrokinase(LK), as a new antithrombotic medicine, has been widely used in prevention and treatment of ischaemic cerebrovascular diseases because of its thrombolytics function. At present, it is usually extracted from the earthworms and is difficult and time-consuming to be purifed. In order to solve these problems, we developed a new method to obtain Lumbrokinase by means of gene-engineering. Lumbrokinase was expressed in E. coli and Pichia pastoris. In this research, cloning and expression of lumbrokinase gene in E. coli and Pichia pastoris were studied .the main results were as follows.In E. coli:The cloned genes can be expressed in different host cells, include E. coli,Bacillus subtilis,yeast,insect cells,cultured mammalian cells,as well as the whole animal. With the different expression systems,it is necessary to build a different expression vectors. The probability that we succeed to express cloned gene in different host cells depends on how much we know about the rules of gene expressing regulation in these system. After long-term research on E. coli,people have understood its genetic and properties very clearly. There are many advantages with the expression system of E. coli,such as simple culture,rapid growth reproduction,and low price. More than 20 years of experience was accumulated in utilizing E. coli to express exogenous gene. The expression level of the exogenous protein in E. coli is much higher than other systems. The Expression quantity of interest protein can be even more than 80% of the total bacterial protein. Thus the protein expression system of E. coli have been widely used.In this study,the expression vector pPelB we used, have a signal peptide, which can make the interest protein secreted into the periplasm cavity of E. coli. There are many advantages of the expression in the periplasm, firstly,it can be effectively concentrated and purified; secondly,The oxidative environment of periplasm is propitious to protein folding by the right way,in the progress of the protein transferring to the cytoplasm,it is easily to occur signal peptide cleavage in vivo and form the correct N-terminal interest protein; finally,protein degradation in the periplasm is not as widespread as in the cytoplasm. So,both prokaryotic and eukaryotic foreign genes have successfully achieved an effective expression in E. coli periplasm.The works we have carried out and the Results: The gene sequence of EPA was cloned by PCR using pSUMO-EPA as template which is constructed by my teacher.The result showed that the fragment was 708bp.The whole gene of EPA was inserted into pPelB vector to construct pPelB-EPA.The recombinant plasmid,pPelB-EPA, was transformed into E. coli Rosetta. The expression of EPA in E. coli was performed by induction with IPTG in OD600=0.6. The results of SDS-PAGE demonstrated that in E.coli Rosetta recombinant EPA protein gene was expressed with the molecular weight about 31KD,which accord with its theoretical molecular weight .The results of Western blot tell us that the expression of interest proteins in E. coli cytoplasm forms inclusion bodies,so that it has no biological activity.In Pichia pastoris:Pichia Pastoris express system which widely used in gene engineering has the properties of simple operation, distinct genetic background, lower cost price. The system has eukaryote's folding and secrete mechanism, such as the post-translational modification process including proteolysis , folding, glycosylation,Moreover it grows rapidly in simple nitritional ingredient, which can accommodate the needs of large-scale industrialization .So we build an expression system of recombinant lumbrokinase and seek to produce recombinant protein with gene- tic engineering technology.The works we have carried out and the Results: The expression vector pPIC9K-EPA was constructed by inserting gene EPA into yeast expression and secretion plasmid pPIC9K.Plasmid pPIC9K-EPA was linearized with BglII and then transformed into Pichiapastoris strain GS115 cell by electroporation method.Phenotypes of transformants were screened in MM and MD plates to ensure the integration of lumbrokinase gene EPA into yeast chromosome DNA.Methanol was added to a final concentration of 0.5%for the expression of recombination protein every 24h to maintain induction.The result of SDS-PAGE showed that the molecular weight of the expression product was about 35KD,in correspondence with the theoretical molecular weight. The results of Western blot tell us that the immunological activities of the expression product was perfect, indicating that EPA was successfully expressed in secretion form in yeast cell.