Spider silk is a proteinaceous fiber with remarkable mechanical properties spun from spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli (E. coli). So far, recombinantly expressed spidroins often form insoluble inclusion bodies (IBs), which will often be dissolved under extremely harsh conditions in a traditional manner, e.g. either 8 mol/L urea or 6 mol/L guanidine hydrochloride, highly risking to poor recovery of bioactive proteins as well as unexpected precipitations during dialysis process. Here, we present a mild solubilization strategy—one-step heating method to solubilize spidroins from IBs, with combining spidroins’ high thermal stability with low concentration of urea. A 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus was expressed in E. coli, and the recombinant proteins were mainly present in insoluble fraction as IBs. The isolated IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method, respectively. The solubilization efficiency of NM IBs in Tris-HCl pH 8.0 containing 4 mol/L urea by one-step heating method was already comparable to that of 7 mol/L urea with using traditional urea-denatured method. The effects of buffer, pH and temperature conditions on NM IBs solubilization of one-step heating method were evaluated, respectively, based on which the recommended conditions are: heating temperature 70–90 °C for 20 min, pH 7.0–10, urea concentration 2–4 mol/L in normal biological buffers. The recombinant NM generated via the one-step heating method held the potential functions with self-assembling into sphere nanoparticles with smooth morphology. The one-step heating method introduced here efficiently solubilizes IBs under relatively mild conditions compared to the traditional ones, which might be important for the downstream applications; however, this protocol should be pursued carefully in terms of urea-induced modification sensitive applications. Further, this method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to other thermal stable proteins.

中文翻译：

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从包涵体有效溶解重组蜘蛛丝蛋白的一步加热策略。

蜘蛛丝是一种蛋白质纤维，具有由蜘蛛丝蛋白（spidroins）纺制而成的出色机械性能。已经在多种异源宿主中成功生产了工程螺旋体，最广泛使用的表达系统是大肠杆菌（E. coli）。到目前为止，重组表达的spidroins通常会形成不溶性包涵体（IBs），它们通常会在极端苛刻的条件下以传统方式溶解，例如8 mol / L尿素或6 mol / L盐酸胍，极有可能导致回收率低生物活性蛋白以及透析过程中的意外沉淀。在这里，我们提出了一种温和的增溶策略-一步加热法来溶解IB中的spidroins，同时将spidroins的高热稳定性与低浓度的尿素结合起来。源自美洲阿文氏菌的小壶腹spidroin的430-aa重组蛋白（称为NM）在大肠杆菌中表达，并且重组蛋白主要以不溶级分形式作为IBs存在。分离的IB分别通过传统的尿素变性方法和一步加热法平行溶解。通过一步加热方法，在含有4 mol / L尿素的Tris-HCl pH 8.0中，NM IBs的增溶效率已与使用传统尿素变性方法的7 mol / L尿素的增溶效率相当。分别评估了缓冲液，pH和温度条件对NM IBs一步加热溶解的影响，基于这些条件，建议的条件为：加热温度70–90°C 20分钟，pH 7.0–10，尿素浓度在普通生物缓冲液中为2–4 mol / L。通过一步加热方法生成的重组NM具有潜在的功能，可以自我组装成具有光滑形态的球形纳米颗粒。与传统方法相比，此处介绍的一步加热方法可以在相对温和的条件下有效地溶解IB，这对于下游应用可能很重要。但是，就尿素诱导的修饰敏感应用而言，应认真遵循该方案。此外，该方法可在较宽的缓冲液，pH和温度条件下应用，从而有可能应用于其他热稳定蛋白。与传统方法相比，此处介绍的一步加热方法可以在相对温和的条件下有效地溶解IB，这对于下游应用可能很重要。但是，就尿素诱导的修饰敏感应用而言，应认真遵循该方案。此外，该方法可在较宽的缓冲液，pH和温度条件下应用，从而有可能应用于其他热稳定蛋白。与传统方法相比，此处介绍的一步加热方法可以在相对温和的条件下有效地溶解IB，这对于下游应用可能很重要。但是，就尿素诱导的修饰敏感应用而言，应认真遵循该方案。此外，该方法可在较宽的缓冲液，pH和温度条件下应用，从而有可能应用于其他热稳定蛋白。