The Preparation Of Insulin Analog

The fast-acting analogs of insulin display faster absorption kinetics and more closely mimic endogenous insulin secretion, compared with human insulin. This thesis is divided to two chapters. Chapter Ⅰ is about the preparation of B22Asp Des-B30Insulin, and the chapter Ⅱ is about the preparation of B27K insulin (the precursor of B27K-DTrI).B22Asp Des-B30Insulin was reported to have monomeric property with50%insulin activity, suggesting that B22Asp Des-B30insulin had potential application in clinic. In our work, B22D Des-B30insulin precursor was prepared by intein mediated expression in E. coli. The expression of B22D Des-B30insulin precursor was200.33mg per liter of culture. Then the B22D Des-B30insulin precursor was harvested by intein splicing. To improve cleavage rate in vitro, pH, temperature, time and the contents of cleavage buffer were used as optimized parameters. As a result, we confirmed that cleavage of the fusion protein was optimal at25℃for6days, in the cleavage buffer (pH5.5) without Urea. At the end of splicing, the pH of cleavage buffer was adjusted to pH8.0, and mixed for another2hours. The B22D Des-B30insulin precursor converted to B22D Des-B30insulin by tryptic hydrolysis and purified by HPLC. The purified B22D Des-B30insulin was analyzed by mass spectrometry. The molecular mass was consistent with the theoretical value (5663). The yield of B22D Des-B30insulin was0.621mg/L.The B27K-DTrI insulin (human insulin with B27-30removed and B27Thr replaced by Lys) was reported to have superior monomeric property with80%insulin activity in vivo. Although it is a new potential fast-acting analog of insulin, the yield of B27K-DTrI insulin is too low to be used in clinic. It was reported that the aggregation of insulin can improve the stability of insulin. So we constructed and expressed the B27K insulin (which is the insulin with the full-length of B strain and B27Thr replaced by Lys) in P. Pastoris. After screening with high concentration of G418and analyzing the results of RT-PCR, seven recombinants with high copies of target gene were obtained. To improve the yield of B27K, the fermentation conditions were optimized. The results of optimal fermentation conditions were as follow:pH5.0,25℃and harvested the cells after108h inducement. Under the same optimal fermentation condition, the yield of B27K reached114mg/L, higher than B27K-DTrI precursor reported before. Then the purified B27K was analyzed by mass spectrometry, the molecular mass was consistent with the theoretical value (6063).