Establishment Of The Purification System For SCT And Optimization Of The Oil Body-based Expression System

Salmon calcitonin (sCT) is first-line drug to treatment of osteoporosis, osteitis deformation and bone pain in clinical application, which has been used more than 30 years. Because the C-terminal of sCT was not amidated in E.coli and yeast expression system, the activity was extremely low (100-200IU/mg). The value of sCT is costly, for example, injection is 12,000yuan/mg and original drug is 18,000-20,000yuan/g. Recently, in order to obtain sCT with high biological activity and low cost people have been trying to find a new way to produce sCT by genetic engineering. The oil body-based expression system in plant is one of optimal system, which is easier to separation and purification of target protein. The recent researches indicated that a part of sCT from oil body-based expression system was amidated, and had higher activities than E.coli and yeast expression system.In this study, we established an optimal purification method based on high-level expression of sCT strains of genetically modified rape. Comprised with many different methods, we found the centrifugal force and time were key factors for acquiring high quality oil body. The optimized protein extraction method of oil body could completely remove impurities, and the obtained oil body was white, no visible particulate impurities and more delicate, which contributed to thrombin digestion in the next experiment. By the comparison of the two methods, extracting oil body digested by thrombin and oil body protein digested by thrombin, we found that the two methods both obtained a target band of 3.45kD, but the latter had more quantity of msCT, indicating cleavage efficiency of this method was higher. The oil body from the seed of transgenic rape was digested by thrombin, and recoveried aqueous phase to freeze drying. We could detect msCT characteristic peak by HPLC and needed a further sequence test.To solve the problems of fromer expression system, we optimized oil body-based expression system as follows: a.) introduced the His-tag;b.) used enterokinase as protease-restricted site instead of thrombin; c.) used GUS gene as a reporter; d.) introduced PHM gene to improve the level of amidated sCT.According to dicotyledons preference codon usage, GC content and protease-restricted site of enterokinase, sCT gene and sesame loeosin gene containing His-tag was synthesized and cloned respectively. Finally, we constructed a plant expression vector pBOsC2301 that contained sesame loeosin-sCT fusion gene triggered by rape oloesin promoter, and integrated it into the genome of rape variety ZHONGSHUANG 4 by Agrobacterium, obtained 14 transgenic plants. PCR analysis indicated that the fusion gene had integrated into the genome of rape and RT-PCR analysis, GUS detection and southern blot showed that the fusion gene had expressed. By SDS-PAGE, we found that the protein band of oil body changed significantly after digestion by enterokinase, indicated that enterokinase was effective to cleave purified oil body. We are purifying the target protein by HPLC and His-tag purificationIn order to improve activity of sCT using PHM gene, we constructed a plant expression vector pDOV2301 that contained soybean loeosin-PHM fusion gene triggered by Arabidopsis oloesin promoter, sesame loeosin-sCT fusion gene triggered by rape oloesin promoter and GUS gene triggered by CaMV 35S promoter, and integrated it into the genome of rape variety ZHONGSHUANG 4 by Agrobacterium, obtained 14 transgenic plants. PCR analysis showed that they were positive.Establishment of the purification system for sCT and optimization of the oil expression system in this study were important to reduce the cost of production and purification...