Yu Wenxi, Yu Maiya, Papasergi-Scott Makaía M, Tall Gregory G
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Protein Expr Purif. 2019 Feb;154:98-103. doi: 10.1016/j.pep.2018.10.002. Epub 2018 Oct 2.
Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activities of Ric-8A are regulated by phosphorylation of five serine and threonine residues within protein kinase CK2 consensus sites. The traditional way that Ric-8A proteins have been purified is from Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells. Endogenous insect cell kinases do phosphorylate the critical regulatory sites of recombinant Ric-8A reasonably well, but there is batch-to-batch variability among recombinant Ric-8A preparations. Additionally, insect cell-production of some Ric-8 proteins with phosphosite alanine substitution mutations is proscribed as there seems to be interdependency of multi-site phosphorylation for functional protein production. Here, we present a method to produce wild type and phosphosite mutant Ric-8A proteins that are fully occupied with bound phosphate at each of the regulatory positions. Ric-8A proteins were expressed and purified from E. coli. Purified Ric-8A was phosphorylated in vitro with protein kinase CK2 and then re-isolated to remove kinase. The phosphorylated Ric-8A proteins were ∼99% pure and the completeness of phosphorylation was verified by chromatography, phos-tag SDS-PAGE mobility shifts, immunoblotting using phospho-site specific antibodies, and mass spectrometry analysis. E. coli-produced Ric-8A that was phosphorylated using this method promoted a faster rate of Gα subunit guanine nucleotide exchange than Ric-8A that was variably phosphorylated during production in insect cells.
抗胆碱酯酶8（Ric-8）蛋白是一种分子伴侣，在生物合成后不久可折叠异源三聚体G蛋白α亚基。Ric-8蛋白还可作为体外鸟嘌呤核苷酸交换因子（GEF），促进Gα亚基的GDP与GTP交换。Ric-8A的GEF和伴侣活性受蛋白激酶CK2共有位点内5个丝氨酸和苏氨酸残基磷酸化的调节。传统上，Ric-8A蛋白是从草地贪夜蛾（Sf9）或粉纹夜蛾（Tni）昆虫细胞中纯化得到的。内源性昆虫细胞激酶确实能较好地磷酸化重组Ric-8A的关键调节位点，但重组Ric-8A制剂之间存在批次间差异。此外，一些具有磷酸化位点丙氨酸替代突变的Ric-8蛋白在昆虫细胞中的生产受到限制，因为功能性蛋白的生产似乎存在多位点磷酸化的相互依赖性。在此，我们提出一种方法来生产野生型和磷酸化位点突变的Ric-8A蛋白，这些蛋白在每个调节位置都完全被结合的磷酸盐占据。Ric-8A蛋白在大肠杆菌中表达和纯化。纯化后的Ric-8A在体外与蛋白激酶CK2一起磷酸化，然后重新分离以去除激酶。磷酸化的Ric-8A蛋白纯度约为99%，通过色谱法、磷标签SDS-PAGE迁移率变化、使用磷酸化位点特异性抗体的免疫印迹以及质谱分析验证了磷酸化的完整性。用这种方法磷酸化的大肠杆菌产生的Ric-8A比在昆虫细胞生产过程中可变磷酸化的Ric-8A促进Gα亚基鸟嘌呤核苷酸交换的速度更快。
