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Association of high pressure and alkaline condition for solubilization of inclusion bodies and refolding of the NS1 protein from zika virus.
BMC Biotechnology ( IF 3.5 ) Pub Date : 2018-12-14 , DOI: 10.1186/s12896-018-0486-2
Cleide Mara Rosa da Silva 1 , Rosa Maria Chura-Chambi 1 , Lennon Ramos Pereira 2 , Yraima Cordeiro 3 , Luís Carlos de Souza Ferreira 2 , Ligia Morganti 1
Affiliation  

BACKGROUND Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein. RESULTS Pressure-treatment (2.4 kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH 8.5, NS1 was refolded and formed soluble oligomers. High (up to 68 mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH 11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods. CONCLUSIONS The present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein.

中文翻译:

高压和碱性条件与包涵体溶解和寨卡病毒NS1蛋白重折叠有关。

背景技术包涵体(IBs)中的蛋白质具有天然的二级结构。但是,广泛用于IB增溶和随后的重折叠的变性浓度的离液剂可展开这些二级结构。离液剂的去除经常引起生物活性蛋白的重新聚集和较差的回收。高静水压(HHP)和碱性pH是两个条件,在低水平的离液剂存在下,已被描述为非变性增溶剂。在本研究中,我们使用zika病毒(ZIKV)非结构1(NS1)蛋白的抗原形式作为模型,评估了HHP和碱性pH值结合对IB增溶的策略。结果NS1-IBs在pH值为11时进行压力处理(2.4 kbar)。0导致低水平的NS1展开并导致IB增溶,主要是变成单体。在pH 8.5透析后,NS1重新折叠并形成可溶性低聚物。在精氨酸(Arg)存在下,通过在pH 11下溶解NS1-IBs可获得高(高达68 mg / L)的NS1浓度,最终产量约为总蛋白含量的80%。事实证明该方法是高效,快速的,不需要进一步的纯化步骤。重新折叠的NS1保留了与抗原特异性抗体(包括ZIKV感染患者血清)反应的生物学特征。与传统的IB增溶-重折叠方法相比,该方法导致增加了约30倍。结论目前的结果代表了通过同时使用HHP和碱性pH来进行创新的非变性蛋白重折叠过程。所报道方法的应用允许在保持蛋白质抗原特性的条件下回收ZIKV NS1。
更新日期:2019-11-01
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