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首页> 外文期刊>Protein Expression and Purification >The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides
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The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides

机译：CNBr裂解反应中的溶剂决定了重组肽生产中使用的酮固醇异构酶融合蛋白的裂解效率

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摘要

Abnormal fragmentation during cyanogen bromide polypeptide cleavage rarely occurs, although parallel side reactions are known to typically accompany normal cleavage. We have observed that cyanogen bromide cleavage of highly hydrophobic fusion proteins utilized for production of recombinant peptides results in almost complete abolishment of the expected reaction products when the reaction is carried out in 70% trifluoroacetic acid. On the basis of mass spectrometric analysis of the reaction products, we have identified a number of fragments whose origin can be attributed to incomplete fragmentation of the fusion protein, and to unspecific degradation affecting the carrier protein. Substituting the solvent in the reaction media with 70% formic acid or with a matrix composed of 6 M guanidinium hydrochloride in 0.1 M HCl, however, was found to alleviate polypeptide cleavage. We have attributed the poor yields of the CNBr cleavage carried out in 70% TFA to the increased hydrophobicity of our particular fusion proteins, and to the poor solubilizing ability of this reaction medium. We propose the utilization of chaotropic agents in the presence of diluted acids as the preferred cyanogen bromide cleavage medium of fusion proteins in order to maximize cleavage efficiency of hydrophobic sequences and to prevent deleterious degradation and structural modifications of the target peptides. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 26]
机译：尽管已知平行的副反应通常伴随正常裂解，但很少发生溴化氰多肽裂解过程中的异常断裂。我们已经观察到，当在70％三氟乙酸中进行反应时，用于生产重组肽的高疏水性融合蛋白的溴化氰裂解会导致预期反应产物几乎完全被消除。在反应产物的质谱分析的基础上，我们确定了许多片段，其起源可归因于融合蛋白的不完全片段化以及影响载体蛋白的非特异性降解。然而，发现在反应介质中用70％甲酸或由6M盐酸胍在0.1M HCl中组成的基质代替溶剂可减轻多肽裂解。我们将在70％TFA中进行的CNBr裂解的收率差归因于我们特定融合蛋白的疏水性增加，以及这种反应介质的增溶能力差。我们提出在稀释的酸存在下利用离液剂作为融合蛋白的优选溴化氰裂解介质，以最大化疏水序列的裂解效率并防止靶肽的有害降解和结构修饰。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：26]

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