Grisshammer R, Duckworth R, Henderson R
Cambridge Centre for Protein Engineering/MRC Centre, U.K.
Biochem J. 1993 Oct 15;295 ( Pt 2)(Pt 2):571-6. doi: 10.1042/bj2950571.
With the goal of obtaining sufficient quantities of seven-helix G-protein-coupled receptors for structural analysis, we have studied the functional expression of a rat neurotensin receptor cDNA in Escherichia coli with and without a signal sequence and as a fusion with the gene coding for maltose-binding protein. The addition of an N-terminal signal peptide resulted in increased expression levels. In vitro translation at a high level revealed that the codon usage of the rat neurotensin receptor cDNA was not critical for overproduction. Expression of neurotensin receptor cDNA fused to the 3' end of the gene encoding maltose-binding protein resulted in a 40-fold increase in neurotensin-binding sites. Binding of [3H]neurotensin to intact bacteria or E. coli membranes was saturable, with a dissociation constant, KD, of 0.23 nM (Bmax. = 450 sites/bacterium or 15 pmol/mg of crude membrane protein). The binding properties of all recombinant receptors presented in this study were similar and corresponded to those of the high-affinity binding sites in rat brain. For immunological detection and future purification of neurotensin receptor, a C-terminal pentahistidine/c-myc tail was introduced. Western-blot analysis revealed the association of neurotensin receptor with E. coli membranes.
为了获得足够数量的七螺旋G蛋白偶联受体用于结构分析，我们研究了大鼠神经降压素受体cDNA在有或没有信号序列的情况下，以及与编码麦芽糖结合蛋白的基因融合时，在大肠杆菌中的功能表达。添加N端信号肽导致表达水平提高。高水平的体外翻译表明，大鼠神经降压素受体cDNA的密码子使用情况对过量生产并不关键。与编码麦芽糖结合蛋白的基因3'端融合的神经降压素受体cDNA的表达导致神经降压素结合位点增加了40倍。[3H]神经降压素与完整细菌或大肠杆菌膜的结合是可饱和的，解离常数KD为0.23 nM（Bmax = 450个位点/细菌或15 pmol/mg粗膜蛋白）。本研究中呈现的所有重组受体的结合特性相似，与大鼠脑中高亲和力结合位点的特性相符。为了进行神经降压素受体的免疫检测和未来的纯化，引入了C端五组氨酸/c-myc尾。蛋白质印迹分析揭示了神经降压素受体与大肠杆菌膜的关联。
