蛋白质功能化的表面有可能开发新的测定法,用于确定潜在化合物的药物样特性并发现G蛋白偶联受体(GPCRs)的特定伴侣。然而,纯化和固定功能性GPCRs的通用方法仍然难以捉摸。为此,我们开发了一种通用且快速的方法来通过硅特异性肽纯化和固定β2-肾上腺素能受体(β2AR)。我们从六种二氧化硅结合肽(minTBP-1,CotB1p,SB7,Car9和Si4-1)通过检查它们对大孔硅胶的亲和力。我们研究了CotB1p标记的β2-肾上腺素受体(β2AR-CotB1p)在不同条件下的吸附和解吸,以提出受体纯化和固定的方案。在优化条件下,β2AR的固定是通过直接浸入带有受体的细胞裂解物与硅胶来实现的。并且通过在洗脱液中包括1-精氨酸/L-赖氨酸来实现受体的洗脱,而没有可证明的污染物。这允许从大肠杆菌(E.coli)裂解物,纯度为95%。通过比较CotB1p策略与典型的共价方法,将固定的受体用作固定相以揭示标签对配体结合输出的影响。沙丁胺醇的KAs,氯丙肾上腺素,妥洛特罗,β2AR-CotB1p柱上的特布他林分别为1.26×106、6.59×106、7.90×106和8.97×105M-1,比Halo-β2AR柱高两个数量级。整个固定在30分钟内完成,无需任何特殊处理。从而提高了确定受体-配体结合参数的准确性。一起来看,CotB1p介导的策略很简单,快速,并普遍用于纯化或固定不稳定的生物分子,如用于分析和生物应用的GPCRs。
Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize β2-adrenergic receptor (β2AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB7, Car9, and Si4-1) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged β2-adrenoceptor (β2AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, β2AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The KAs of salbutamol, chlorprenaline, tulobuterol, and terbutaline on β2AR-CotB1p column were 1.26 × 106, 6.59 × 106, 7.90 × 106, and 8.97 × 105 M-1 respectively, which were two orders of magnitude higher than those on the Halo-β2AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.