Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, 3010, Australia.
Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, The University of Melbourne, Parkville, VIC, 3010, Australia.
Sci Rep. 2017 Apr 24;7(1):1094. doi: 10.1038/s41598-017-01227-z.
G-protein coupled receptors are the largest family of integral membrane proteins found within the human genome. They function as receptors and modulators to a wide range of ligands and responses which are crucial for human health. GPCR study, specifically the investigation of structure and interaction to cognate ligands, is of high priority. Limitations for structural study can be traced in part, to obtaining suitable quantities of recombinant protein. We sought to address the limitations of traditional recombinant technologies by utilising an Escherichia coli based cell-free protein synthesis (CFPS) approach for production of a thermostable neurotensin receptor 1 (en2NTS). Initial results were promising, with a high amount (up to 2 mg/mL) of en2NTS produced, that had attained correct secondary structure. Meanwhile, concurrent experiments indicated that CFPS produced en2NTS showed non-competitive binding to the peptide ligand neurotensin8-13 when compared to E. coli produced en2NTS. H-C HMQC SOFAST NMR spectra were indicative of disrupted tertiary structure for CFPS produced CH-methionine labelled en2NTS. The results obtained, indicate CFPS produced en2NTS is not forming a discrete tertiary structure and that further development of the CFPS technique needs to be carried out.
G 蛋白偶联受体是人类基因组中发现的最大的整合膜蛋白家族。它们作为受体和调节剂，作用于广泛的配体和反应，对人类健康至关重要。GPCR 研究，特别是对结构和与同源配体相互作用的研究，是当务之急。结构研究的局限性部分可以追溯到获得足够数量的重组蛋白。我们试图通过利用基于大肠杆菌的无细胞蛋白合成 (CFPS) 方法来生产耐热的神经降压素受体 1 (en2NTS)，来解决传统重组技术的局限性。最初的结果很有希望，产生了大量（高达 2mg/mL）的 en2NTS，其具有正确的二级结构。同时，同时进行的实验表明，与大肠杆菌产生的 en2NTS 相比，CFPS 产生的 en2NTS 对肽配体神经降压素 8-13 表现出非竞争性结合。H-C HMQC SOFAST NMR 谱表明 CFPS 产生的 CH-甲硫氨酸标记的 en2NTS 的三级结构被破坏。获得的结果表明，CFPS 产生的 en2NTS 没有形成离散的三级结构，需要进一步开发 CFPS 技术。
