Departments of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
Cell Biology and Biochemistry, and the Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
Methods Mol Biol. 2022;2507:337-358. doi: 10.1007/978-1-0716-2368-8_18.
Normal functions of cell-surface proteins are dependent on their proper trafficking from the site of synthesis to the cell surface. Transport proteins mediating solute transfer across the plasma membrane constitute an important group of cell-surface proteins. There are several diseases resulting from mutations in these proteins that interfere with their transport function or trafficking, depending on the impact of the mutations on protein folding and structure. Recent advances in successful treatment of some of these diseases with small molecules which correct the mutations-induced folding and structural changes underline the need for detailed structural and biophysical characterization of membrane proteins. This requires methods to express and purify these proteins using heterologous expression systems. Here, using the solute carrier (SLC) transporter NaCT (Na-coupled citrate transporter) as an example, we describe experimental strategies for this approach. We chose this example because several mutations in NaCT, distributed throughout the protein, cause a severe neurologic disease known as early infantile epileptic encephalopathy-25 (EIEE-25). NaCT was modified with various peptide tags, including a RGS-His, a Twin-Strep, the SUMOstar domain, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. When transiently expressed in HEK293 cells, recombinant NaCT proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited citrate transport activity similar to the nontagged protein. Surface NaCT expression was enhanced by the presence of SUMOstar on the N-terminus. The dual-purpose peptide epitopes RGS-His and Twin-Strep facilitated detection of NaCT by immunohistochemistry and western blot and may serve useful tags for affinity purification. This approach sets the stage for future analyses of mutant NaCT proteins that may alter protein folding and trafficking. It also demonstrates the capability of a transient mammalian cell expression system to produce human NaCT of sufficient quality and quantity to augment future biophysical and structural studies and drug discovery efforts.
细胞表面蛋白的正常功能依赖于它们从合成部位正确转运到细胞表面。介导溶质跨质膜转移的转运蛋白构成了细胞表面蛋白的一个重要群体。有几种疾病是由于这些蛋白质的突变引起的，这些突变干扰了它们的运输功能或转运，具体取决于突变对蛋白质折叠和结构的影响。最近，一些使用小分子成功治疗这些疾病的进展表明，需要对膜蛋白进行详细的结构和生物物理特性分析。这需要使用异源表达系统来表达和纯化这些蛋白质的方法。在这里，我们以溶质载体 (SLC) 转运体 NaCT（Na 偶联柠檬酸转运体）为例，描述了这种方法的实验策略。我们选择这个例子是因为 NaCT 中的几个突变，分布在整个蛋白质中，导致一种严重的神经系统疾病，称为早发性婴儿癫痫性脑病-25（EIEE-25）。NaCT 被各种肽标签修饰，包括 RGS-His、Twin-Strep、SUMOstar 结构域和增强型绿色荧光蛋白（EGFP），每个标签单独或组合使用。当瞬时表达在 HEK293 细胞中时，重组 NaCT 蛋白经历了复杂的糖基化，与质膜分隔开，并表现出与未标记蛋白相似的柠檬酸转运活性。N 端存在 SUMOstar 可增强表面 NaCT 的表达。双用途肽表位 RGS-His 和 Twin-Strep 有助于通过免疫组织化学和 Western blot 检测 NaCT，并且可能作为亲和纯化的有用标签。这种方法为未来分析可能改变蛋白质折叠和转运的突变型 NaCT 蛋白奠定了基础。它还展示了瞬时哺乳动物细胞表达系统生产足够质量和数量的人类 NaCT 的能力，以增强未来的生物物理和结构研究以及药物发现工作。
