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首页> 外文期刊>Biotechnology Progress >Effects of Citraconylation on Enzymatic Modification of Human Proinsulin Using Trypsin and Carboxypeptidase B
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Effects of Citraconylation on Enzymatic Modification of Human Proinsulin Using Trypsin and Carboxypeptidase B

机译：柠檬酸基化对胰蛋白酶和羧肽酶B对人胰岛素原酶促修饰的影响

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摘要

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.
机译：胰岛素是一种多肽激素，由胰腺的β细胞产生，可​​控制人体的血糖水平。使用胰蛋白酶和羧肽酶B对人胰岛素原进行酶促修饰通常会导致胰岛素衍生物大量积累，从而导致纯化过程更加复杂。为了减少主要衍生物des-苏氨酸人胰岛素，开发了一种简单的方法，包括酶促修饰过程中的柠康酰化和去柠康酰化。将每克蛋白质3.0 g柠康酸酐添加到反应溶液中导致人胰岛素原中赖氨酸残基的柠康酰化，并使相对苏氨酸胰岛素相对含量从13.5％降低至1.0％。柠康酰化的胰岛素原经过酶促水解后，可以将赖氨酸残基的100％进行去磺酰化，并通过在25°C下将pH调节至2-3来恢复。在重组大肠杆菌中表达的过氧化氢添加和胰岛素原的瓜皮酰化的结合显着地将胰岛素的转化率从52.7提高到77.7％。因此，赖氨酸残基的甲磺酰化作用阻止了胰蛋白酶对人胰岛素的意外裂解，使去苏氨酸胰岛素的形成减至最少，从而提高了活性胰岛素的产量。

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