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首页> 外文期刊>Journal of Chemical Technology & Biotechnology >Solubilization at high pH results in improved recovery of proteins from inclusion bodies of E. coli
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Solubilization at high pH results in improved recovery of proteins from inclusion bodies of E. coli

机译：在高pH下溶解可改善从大肠杆菌包涵体中回收蛋白质的能力

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BACKGROUND: Solubilization of inclusion bodies using high concentration of chaotropes followed by suboptimal refolding results in lower recovery of bioactive proteins from E. coli. Growing evidence indicates that aggregation of expressed proteins into inclusion bodies is molecular specific in nature and proteins in inclusion bodies have native-like secondary structure. Protecting these pre-existing native-like secondary structures of proteins using mild solubilization conditions is one of the key steps in improved recovery of bioactive protein from inclusion bodies of E. coli. RESULTS: Inclusion bodies of seven proteins (hGH, oGH, Pyk, PKS, aldolase, enolase and superoxide dismutase) were solubilized at pH 12 in the presence of 2mol L-1 urea and were subsequently refolded and purified. Enrichment of inclusion bodies after cell lysis, mild solubilization at alkaline pH and pulsatile refolding resulted in lower aggregation of protein during refolding and improved the final recovery of protein from inclusion bodies of E. coli. The efficiency of protein recovery from inclusion bodies was 30-40% for most of the proteins. This was almost two times higher than that achieved when inclusion bodies were solubilized in 8 mol L-1 urea and subsequently refolded. Purified hGH and Pyk were functionally active in respective in vitro assays. CONCLUSION: Non-denaturing solubilization of inclusion body proteins at alkaline pH is a viable alternative to the use of high molar concentration chaotropes for efficient recovery of inclusion body proteins. Mild solubilization and pulsatile refolding contribute synergistically to reduce the extent of aggregation during refolding and result in high throughput recovery of bioactive proteins from inclusion bodies of E. coli. (C) 2008 Society of Chemical Industry.
机译：背景：使用高浓度的离液剂溶解包涵体，然后进行次优折叠，导致大肠杆菌中生物活性蛋白的回收率降低。越来越多的证据表明，表达的蛋白质聚集到包涵体中本质上是分子特异性的，包涵体中的蛋白具有天然的二级结构。使用温和的增溶条件来保护这些先前存在的类似天然蛋白质的二级结构是改善从大肠杆菌包涵体中回收生物活性蛋白质的关键步骤之一。结果：在2mol L-1尿素的存在下，在pH 12下可溶解7种蛋白质（hGH，oGH，Pyk，PKS，醛缩酶，烯醇酶和超氧化物歧化酶）的包涵体，然后对其进行折叠和纯化。细胞裂解后包涵体的富集，在碱性pH值下的适度溶解和搏动性重折叠导致蛋白质在重折叠过程中的聚集度降低，并改善了从大肠杆菌包涵体中最终蛋白质的回收率。对于大多数蛋白质，从包涵体中回收蛋白质的效率为30-40％。这几乎是将包涵体溶解在8 mol L-1尿素中并随后重新折叠时所获得的两倍。纯化的hGH和Pyk在各自的体外测定中具有功能活性。结论：在碱性pH条件下，包涵体蛋白的非变性增溶是替代高摩尔浓度离液剂有效回收包涵体蛋白的一种可行方法。轻度的溶解和搏动性折叠起协同作用，以减少重新折叠过程中的聚集程度，并导致从大肠杆菌包涵体中回收高生物活性蛋白。 （C）2008年化学工业协会。

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