Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies
Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies
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开发从经典包涵体中回收多种重组蛋白的单一方法的比较研究
DOI：
10.1007/s00253-018-8754-6
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发表时间：
2018-03-01
影响因子：
5
通讯作者：
Zhong, Mintao
中科院分区：
文献类型：
--
作者：
Padhiar, Arshad Ahmed;Chanda, Warren;Zhong, Mintao
包涵体的形成被认为是外源蛋白在细菌宿主中表达的阿喀琉斯之踵。已经开发了广泛的技术来从IB中回收具有生物化学挑战性的蛋白质。然而，发现即使从相同的蛋白质家族获得活性状态也是独立于单一建立的方法的。在这里，我们提出了一种新的策略，从苛刻的IB重组蛋白的广泛的亚类的回收。我们发现，如果包涵体是粗糙的，那么用于减少IB形成和产生可溶性蛋白的许多方法及其组合是无效的。另一方面，使用温和增溶缓冲液的不同实践能够完全增溶IB，但活性蛋白的回收需要大量筛选重折叠缓冲液。结合先前报道的温和增溶技术，我们提出了一种改进的方法，该方法包括低肌氨酸浓度，范围为0.05至0.1%，加上缓慢冷冻（− 1 °C/min）和快速解冻（室温），从而获得更大的溶解度和溶解蛋白的完整性。采用单一缓冲液稀释法恢复重组蛋白的活性。结果表明，回收的蛋白质的活性显着高于传统的增溶/复性方法。通过所述方法溶解IB被证明在性质上更温和，这恢复了IB内蛋白质的天然样构象。
The formation of inclusion bodies (IBs) is considered as an Achilles heel of heterologous protein expression in bacterial hosts. Wide array of techniques has been developed to recover biochemically challenging proteins from IBs. However, acquiring the active state even from the same protein family was found to be an independent of single established method. Here, we present a new strategy for the recovery of wide sub-classes of recombinant protein from harsh IBs. We found that numerous methods and their combinations for reducing IB formation and producing soluble proteins were not effective, if the inclusion bodies were harsh in nature. On the other hand, different practices with mild solubilization buffers were able to solubilize IBs completely, yet the recovery of active protein requires large screening of refolding buffers. With the integration of previously reported mild solubilization techniques, we proposed an improved method, which comprised low sarkosyl concentration, ranging from 0.05 to 0.1% coupled with slow freezing (− 1 °C/min) and fast thaw (room temperature), resulting in greater solubility and the integrity of solubilized protein. Dilution method was employed with single buffer to restore activity for every sub-class of recombinant protein. Results showed that the recovered protein’s activity was significantly higher compared with traditional solubilization/refolding approach. Solubilization of IBs by the described method was proved milder in nature, which restored native-like conformation of proteins within IBs.