site > International journal > (2009) Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B.

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작성일 : 12-07-17 18:03

(2009) Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B.

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http://onlinelibrary.wiley.com/doi/10.1002/btpr.195/abstract [763]

Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B.

Young-Jin Son, Chang-Kyu Kim, Young-Bong Kim, Dae-Hyuk Kweon, Yong-Cheol Park* and Jin-Ho Seo*
Biotechnology Progress 25(4): 1064-1070, 2009.08.01.
(SCI I/F: 2.340)

Insulin is a polypeptide hormone which is produced by the β-cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des-threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des-threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2–3 at 25 °C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des-threonine insulin and hence increased the production yield of active insulin.

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