원문정보
초록
영어
A pPT-BRpi expression vector for the production of human insulin was constructed and transformed into E. coli JM109. E. coli JM109/pPT-BRpi has the P2 promoter, lac operator, and ColE1 origin of replication. E. coli JM109/pPT-BRpi was cultured by a three-step temperature shift fermentation method. The induction of human preproinsulin expression was made by a temperature shift from 30℃ to 37℃. The dry cell weight and preproinsulin content in the three-step temperature shift fermentation with E. coli JM109/pPT-BRpi were higher than those of the constant temperature fermentation. The three-step temperature shift fermentation gave the final dry cell weight of 45.5 g/L and expression content of 69.0%.
Preproinsulin was expressed as inclusion body in recombinant E. coli. The inclusion bodies
were solubilized with β-mercaptoethanol and refolded to restore biological activity. The folded
preproinsulin was transformed into human insulin by the enzyme reactions with trypsin and
carboxypeptidase B. The most cumbersome problem with conventional downstream processes
was the formation of many human insulin derivatives at the enzymatic modification step. Desthreonine (B30) insulin and insulin fragments were synthesized during the enzyme reaction.
Des-threonine (B30) human insulin which is human insulin without B-30 threonine was formed
when the C-terminal of B29-lysine of preproinsulin was cleaved by trypsin. The insulin
fragments were formed by β-mercaptoethanol during the enzyme reaction as well. To minimize
the formation of human insulin fragments by β-mercaptoethanol, hydrogen peroxide was used
during the enzyme reaction with trypsin and carboxypeptidase B. To block the formation of desthreonine human insulin, citraconic anhydride was used during the enzyme reaction.
Combination of H2O2 addition and citraconylation improved remarkably the insulin production yield to 50%. The overall yield of the purified human insulin from the enzyme reaction with
citraconylated preproinsulin to crystallization was about 67%. In a 200 L fed-batch fermentation,
0.485 g human insulin could be produced per 1 L fermentation broth at cell concentration of
45.5 g dry cell weight/L. The purity of purified human insulin was above 98.5%.