Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4B^G12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein–ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4B^G12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4B^G12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20 °C. Native Kras4B^G12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4B^G12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4B^G12V. The native Kras4B^G12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system.

中文翻译：

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pET SUMO蛋白表达系统在大肠杆菌中的应用：天然Kras4BG12V癌蛋白的克隆，表达，纯化和表征。

作为细胞生长和存活的重要调节剂，Kras4B的甘氨酸12残基点突变为缬氨酸（V）使Kras4B ^G12V致癌。Kras4B重组蛋白被用作诱饵，以捕鱼其潜在的配体，以尝试对该癌蛋白进行药物处理，并在蛋白质-配体相互作用研究中验证其与药理相关的配体。然而，Kras4B重组蛋白的合成是有挑战性的，因为据报道其易于聚集到细菌宿主中的包涵体中，导致重组蛋白的产率很低。在这里，我们描述了一种新的方法，通过使用pET SUMO蛋白表达系统作为解决包涵体形成的方法来生产天然Kras4B ^G12V蛋白。克拉斯^{4B G12V}将癌基因克隆到pET SUMO载体中，然后在20°C下在大肠杆菌中化学诱导12 h蛋白表达。天然Kras4B ^G12V蛋白是通过一系列蛋白纯化步骤生产的，这些步骤包括固定的镍离子亲和柱色谱，SUMO融合蛋白和多组氨酸标签去除，以及尺寸排阻柱色谱。通过免疫印迹分析验证了纯化的Kras4B ^G12V蛋白的身份。纯化的蛋白质表现出自我二聚化，表明纯化的蛋白质在结构上类似于Kras4B。它与Kras4B的已知结合剂4,6-二氯-2-甲基-3-氨基乙基-吲哚（DCAI）的物理相互作用证实了纯化的蛋白质与Kras4B ^G12V相同。通过使用pET SUMO蛋白表达系统成功地大量纯化了天然Kras4B ^G12V蛋白。