The Study Of Praperation Of Calcitonin And Insulin Analogs By Impact-Twin System

Biologically active peptides are compounds with good activity and high utilization. Currently, a variety of biologically active peptides have been used as drugs for clinical treatment. In this thesis, the biological preparation of human calcitonin analogs and the refolding method of MIP (monomeric B27Lys destripeptide insulin precursor) fusion protein inclusive bodies were investigated, respectively.In the first part, the cyclic and linear human calcitonin analogs were prepared. Nowadays, osteoporosis has become a health issue worldwide. The global incidence rate is more than25%, which has become the7th common and frequently-occurring disease. Calcitonin is one of the most effective drugs for treating osteoporosis in clinical application. Because of the limited sources of human calcitonin, salmon calcitonin is often used for clinics. However salmon calcitonin has immunogenicity. Therefore, the improvement of the method for preparation of human calcitonin needs to be studied. The physiological activity of calcitonin is closely related to its C-terminal amidated structure. Thus, preparation of human calcitonin by genetic engineering must be amidated in the C-terminal in vitro, which makes the preparation process more complicated and costly. Recent studies indicate that the active peptides with C-terminal amidation can be obtained by cyclization which is formed through linking between head and tail. They show the same activity as linear peptides. This finding solves the problem of C-terminal amidation, and also cyclic peptide has a more stable skeletal structure and functional activity. In this study, the IMPACT-TWIN (Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein) expression system in E.coli was employed to obtain linear peptides or cyclic peptides by different cloning methods. After chitin affinity purification and intein cleavage, polypeptide of amino acid residues without redundancy could be obtained. The whole preparation process has the advantages of fewer steps and less time. In this study, two kinds of human calcitonin analogs were prepared. Human calcitonin analog chCalcitonin (AAP) was a cyclic analog which is lack of the last two amino acid of wild type. Human calcitonin analog hCalcitonin is the full-length human calcitonin.18.5mg/L cyclic human calcitonin analog chCalcitonin (△AP) was obtained using IMPACT-TWIN expression system. Its molecular weight was consistent with the theoretical values by identification of mass spectrometry. Regarding human calcitonin analog hCalcitonin, we optimized the induction time, temperature, IPTG concentration and the medium. It showed that the optimal condition for the expression of he was16℃,16h,0.05mM IPTG.In the second part, the refolding method of monomeric B27Lys destripeptide insulin precursor MIP fusion protein inclusive bodies was investigated. The fast-acting insulin analogs display faster absorption kinetics compared with human insulin and thus more closely mimic endogenous secreted insulin. The monomeric B27Lys destripeptide insulin (B27K-DTrI) is a kind of fast-acting insulin analog with an activity of80%of wild type insulin and has potential clinical values. The monomeric B27Lys destripeptide insulin precursor (MIP) fusion protein often exists in the form of inclusion bodies in the E. coli expression system. Therefore, improvement of the efficiency refolding of recombinant MIP is a key step to increase the yield of active B27K-DTrI. In this work, we compared the refolding efficiency of recombinant MIP by three methods (dialysis, dilution and size exclusion chromatography). The results showed that size exclusion chromatography was the best, which had a refolding efficiency of43.6%. On the basis of the study above, the factors in the refolding buffer were optimized. The content of Arg, ratios of GSSG: GSH and PEG:Pro were chosen as three factors to design the orthogonal experiment. By range and factorial analysis, the results showed that the optimal condition for refolding was2mM GSH,0.4mM GSSG,0.8M Arg, PEG:Pro=2:1. Under the optimal condition, experiment was performed to confirm the result. It showed that refolding efficiency was72%and1.56mg/L of MIP were obtained. The refolding efficiency and productivity were two times higher than those before optimization. The result basically achieved the optimization goal.