胰岛素类似物的生物制备.pdfVIP

a iK O lL C Tr O R f liTT 胰 岛 素类 似 物 的生物 制 济 T H E P R E P A R A T IO N O F IN S U L IN A N A L O G A B S T R A C T T he fast-acting analogs of insu lin d isp lay faster absorption k inetics and m ore closely m im ic endogenous in su lin secretion, com pared w ith hum an insu lin . Th is thesis is d ivided to tw o chapters. C hapter I is about the preparation of B 22 A sp D es-B 30 Insu lin, and the chapter II is about the preparation of B 2 7K insu lin (the precursor of B 27K -D T rI). B 22 A sp D es-B 30 Insulin w as reported to have m onom eric property w ith 50% insulin activity, suggesting that B 22 A sp D es-B 30 insulin had potential app lication in clin ic. In our w ork, B 22D D es-B 30 insu lin precursor w as prepared by intein m ed iated expression in E . coli. T he expression of B 22D D es-B 30 insu lin precursor w as 200.33 m g p er liter of culture. T hen the B 22D D es-B 30 insu lin precursor w as harvested by intein sp licing. To im prove cleavage rate in v itro, pH ， tem perature, tim e and the contents of cleavage buffer w ere used as optim ized param eters. A s a resu lt, w e con firm ed that cleavage of th e fusion protein w as optim al at 25 。C for 6 day s, in the cleavage buffer (pH 5.5) w ithout U rea. A t the end of splicing, the pH o f cleavage buffer w as adjusted to pH 8.0, and m ixed for another 2 hours. T he B 22D D es-B 30 insu lin precursor converted to B 22D D es-B 30 insu lin by tryptic hydro lysis and purified by H P L C . T h e purified B 22D D es-B 30 insu lin w as analyzed by m ass sp ectrom etry . T he m o lecu lar m ass w as con sistent w ith the th eoretical value (5663). T he y ield of B 22 D D es-B 30 insu lin w as 0.62 1 m g/L . T he B 27K -D T rI insu lin (hum an insulin w ith B 27-30