System Optimization Of E. Coli To Produce Pharmaceutical Proteins

Genetic engineering pharmaceutical refers to the use of recombinant technology to overexpress physiological substances in bacteria,yeast and animal cells. These physiological substances include proteins,monoclonal antibodies,cytokines and other new types of drugs which are difficult to prepare using traditional methods. Genetic engineering pharmaceutical made a great impact on the pharmaceutical industry.It is playing an increasing important role in the prevention,diagnosis,control and eradication of diseases.The progress of genetic engineering pharmaceutical will make for the protection of human health and the protraction of the life course.Erythropoietin(EPO)is a model of successful development of the genetic engineering pharmaceutical.Formerly erythropoietin was obtained from the urea of anemia patients and the blood of sheep.The yield was very low and the production was extremely unstable.Its physico-chemical and biological properties were difficult of determine.These disadvantages block its large-scale application.The price of recombinant human erythropoietin(rhEPO)is much lower and curative effect is better in the therapy of renal anemia.Interferon-β(IFN-β)is a glycoprotein generated from fibroblast after affected by the virus or IFN.It is used for the treatment of viral diseases and tumor.In order to obtain a large number of cheap protein drugs,our experiment is designed to express these protein drugs in Escherichia coli expression system.We obtained the EPO and IFN-βgenes by chemical synthesis method for the elimination of the rare codons of EPO and IFN-βnative gene.The two genes were connected to expression vector pET series.Then the vectors were transformed into the host E.coli BL21(DE3).Orthogonal experimental design was used to identify the influence of expression conditions.The yield of target protein is up to 30%after the induction of engineering strain. The result of orthogonal experimental design shows that the best induction condition is 0.8 M IPTG,OD₅₅₀=0.9,4h induction hours. The experimental results show that,we have succeed in constructing stable expression strains of erythropoietin and IFN-β,the strains can be used to produce vast and cheap proteins for the next phase of modifying the native proteins.