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首页> 外文期刊>Biotechnology Progress >Cell-Free Protein Synthesis and Purification of Human Dopamine D2 Receptor Long Isoform
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Cell-Free Protein Synthesis and Purification of Human Dopamine D2 Receptor Long Isoform

机译：人多巴胺D2受体长异构体的无细胞蛋白质合成与纯化

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The human dopamine D2 receptor long isoform (D2L) has significant implications in neurological and neuropsychiatric disorders such as Parkinson s disease and schizophrenia. Detailed structural knowledge of this receptor is limited owing to its highly hydrophobic nature, which leads to protein aggregation and host toxicity when expressed in cellular systems. The newly emerging field of cell-free protein expression presents numerous advantages to overcome these challenges. This system utilizes protein synthesis machinery and exogenous DNA to synthesize functional proteins outside of intact cells. This study utilizes two different cell-free systems for the synthesis of human dopamine D2L receptor. These include the Escherichia coli lysate-based system and the wheat-germ lysate-based system. The bacterial cell-free method used pET 100ID-TOPO vector to synthesize hexa-histidine-tagged D2L receptor using a dialysis bag system; the resulting protein was purified using nickel-nitrilo-triacetic acid affinity resin. The wheat germ system used pEU-glutathione-S-transferase (GST) vector to synthesize GST-tagged D2L receptor using a bilayer translation method; the resulting protein was purified using a GST affinity resin. The presence and binding capacity of the synthesized D2L receptor was confirmed by immunoblotting and radioligand competition assays, respectively. Additionally, in-gel protein sequencing via Nano LC-MSIMS was used to confirm protein synthesis via the wheat germ system. The results showed both systems to synthesize microgram quantities of the receptor. Improved expression of this highly challenging protein can improve research and understanding of the human dopamine D2L receptor.
机译：人多巴胺D2受体长异构体（D2L）在神经系统疾病和神经精神疾病（如帕金森氏病和精神分裂症）中具有重要意义。由于该受体的高度疏水性，因此其详细的结构知识受到限制，当在细胞系统中表达时，这会导致蛋白质聚集和宿主毒性。无细胞蛋白质表达的新兴领域展现了克服这些挑战的众多优势。该系统利用蛋白质合成机制和外源DNA在完整细胞外部合成功能性蛋白质。这项研究利用两种不同的无细胞系统合成人多巴胺D2L受体。这些包括基于大肠杆菌裂解物的系统和基于小麦胚芽裂解物的系统。无细菌方法是使用pET 100ID-TOPO载体通过透析袋系统合成六组氨酸标记的D2L受体。用镍-三硝基三乙酸亲和树脂纯化得到的蛋白质。小麦胚芽系统使用pEU-谷胱甘肽-S-转移酶（GST）载体通过双层翻译方法合成GST标签的D2L受体。使用GST亲和树脂纯化得到的蛋白质。分别通过免疫印迹和放射性配体竞争测定法确认了合成的D2L受体的存在和结合能力。另外，通过Nano LC-MSIMS进行的凝胶内蛋白质测序被用于确认通过小麦胚芽系统的蛋白质合成。结果表明，两种系统都能合成微克量的受体。这种具有高度挑战性的蛋白质的改进表达可以改善对人类多巴胺D2L受体的研究和了解。

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