Brown Sherlene L, Ren Yuan, Suter Daniel M, Mattoo Seema
Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Bio Protoc. 2021 Sep 20;11(18):e4158. doi: 10.21769/BioProtoc.4158.
Cortactin is an actin-binding protein that regulates processes like cell migration, endocytosis, and tumor cell metastasis. Although cortactin is associated with actin-cytoskeletal dynamics in non-neuronal cells and cell-free systems, the exact mechanisms underlying its fundamental roles in neuronal growth cones are not fully explored. Recent reports show that Src2 tyrosine kinase induces phosphorylation of cortactin as a mechanism to control lamellipodia protrusion and filopodia formation in cultured bag cell neurons ( He , 2015 ; Ren , 2019 ). In order to provide evidence for Src2-mediated phosphorylation of cortactin, we developed a robust and cost-effective method for the efficient expression and purification of cortactin and Src2 kinase that can be used for biochemical studies including phosphorylation assays. By co-purifying cortactin and Src kinase with a phosphatase (YopH) from , we eliminated the problem of non-specific phosphorylation of induced proteins by bacterial kinases and also reduced costs by bypassing the need for commercial enzymatic treatments. This protocol is reproducible and can be modified to produce homogenous non-phosphorylated proteins during recombinant protein expression in .
皮层肌动蛋白结合蛋白是一种肌动蛋白结合蛋白，可调节细胞迁移、内吞作用和肿瘤细胞转移等过程。尽管皮层肌动蛋白结合蛋白在非神经元细胞和无细胞系统中与肌动蛋白细胞骨架动力学有关，但其在神经元生长锥中发挥基本作用的确切机制尚未得到充分探索。最近的报告表明，Src2酪氨酸激酶诱导皮层肌动蛋白结合蛋白的磷酸化，作为控制培养的袋状细胞神经元中片状伪足突出和丝状伪足形成的一种机制（He，2015；Ren，2019）。为了提供Src2介导的皮层肌动蛋白结合蛋白磷酸化的证据，我们开发了一种强大且经济高效的方法，用于高效表达和纯化皮层肌动蛋白结合蛋白和Src2激酶，可用于包括磷酸化测定在内的生化研究。通过将皮层肌动蛋白结合蛋白和Src激酶与来自的磷酸酶（YopH）共纯化，我们消除了细菌激酶对诱导蛋白进行非特异性磷酸化的问题，并且通过绕过对商业酶处理的需求降低了成本。该方案具有可重复性，并且可以在重组蛋白表达过程中进行修改以产生均匀的非磷酸化蛋白。
