Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and β-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6_His/EK, and GRK6_His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His₁₀ affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6_His/EK, and GRK6_His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6_His/TEV with the His₁₀ tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.

人 G 蛋白偶联受体激酶 6 (GRK6) 属于 G 蛋白偶联受体激酶家族的 GRK4 激酶亚家族，该家族由 GRK1、GRK2 和 GRK4 组成。这些激酶磷酸化配体激活的 G 蛋白偶联受体 (GPCR)，驱动异源三聚体 G 蛋白偶联、GPCR 脱敏和 β-抑制蛋白募集。该反应系列介导细胞存活、增殖、迁移和趋化性的细胞信号通路。GRK6 是多发性骨髓瘤中的激酶靶标，因为与上皮细胞相比，它在骨髓瘤细胞中高度表达，并且在介导 T 和 B 淋巴细胞的趋化反应中具有重要作用。为了支持基于结构的药物设计，我们描述了三种人类 GRK6 结构，GRK6、GRK6 _His/EK和 GRK6 _His/TEV，专为在草地贪夜蛾Sf9 昆虫细胞中表达蛋白质而设计。第一个构建体不包含任何纯化标签，而其他两个构建体包含 His ₁₀亲和标签，这增加了纯化产量。我们在此报告，GRK6 的所有三种构建体在 Sf9 昆虫细胞中均过表达，并在适合 GRK6 与潜在抑制剂共结晶的水平上纯化至均质。纯化的 GRK6、GRK6 _His/EK和 GRK6 _His/TEV的产量分别为每升细胞培养物 0.3 mg、0.8 mg 和 0.7 mg。此外，我们已经证明 GRK6 _His/TEV与 His ₁₀如通过动态光散射测量观察到的，去除的标签是高度均匀的和单分散的，并且如圆二色光谱所显示的那样主动折叠。所描述的方法将支持针对多发性骨髓瘤的其他疗法的基于结构的开发。