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首页> 外文期刊>Protein Expression and Purification >Expression, purification, and PC1-mediated processing of (H10D, P28K, and K29P)-human proinsulin
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Expression, purification, and PC1-mediated processing of (H10D, P28K, and K29P)-human proinsulin

机译：（H10D，P28K和K29P）-人胰岛素原的表达，纯化和PC1介导的加工

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摘要

Our previous methods for the generation of recombinant human proinsulin were inadequate in terms of reproducibility and yield. In addition, it was difficult to perform structure/function studies on proinsulin because of its tendency to form hexamers. We have developed an improved procedure, which overcomes many of the technical purification problems, and results in a potentially monomeric version of modified proinsulin. Inclusion bodies were prepared using a commercial bacterial lysis solution. The inclusion bodies were solubilized and the fusion protein's affinity tag was removed by chemical cleavage. The polypeptide was then reduced and transferred into a refolding buffer. Following an overnight incubation, only a single form of proinsulin was detected using analytical reversed-phase high-performance liquid chromatography. The refolded (H10D, P28K, and K29P)-human proinsulin (DKP-hPI) was subjected to a final purification step using reversed-phase chromatography. The method is reproducible and produces milligram quantities of purified DKP-hPI from a single liter of bacterial culture. The final product is greater than 95% pure and is suitable for use as a substrate for the propeptide convertase PC1. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 26]
机译：就可重复性和产量而言，我们以前的重组人胰岛素原的产生方法是不充分的。另外，由于胰岛素原倾向于形成六聚体，因此难以进行胰岛素原的结构/功能研究。我们开发了一种改进的方法，该方法克服了许多技术纯化问题，并导致了改性胰岛素原的潜在单体形式。使用商业细菌裂解液制备包涵体。溶解包涵体，并通过化学切割去除融合蛋白的亲和标签。然后将多肽还原并转移至重折叠缓冲液中。过夜孵育后，使用分析型反相高效液相色谱法仅检测到单一形式的胰岛素原。将重折叠的（H10D，P28K和K29P）-人胰岛素原（DKP-hPI）进行最后的纯化步骤，使用反相色谱法。该方法具有可重现性，可从一升细菌培养物中产生毫克量的纯化DKP-hPI。最终产物的纯度大于95％，适合用作前肽转化酶PC1的底物。 （C）2002 Elsevier Science（美国）。版权所有。 [参考：26]

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