Serfling Robert, Seidel Lisa, Böttke Thore, Coin Irene
Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig.
Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig;
J Vis Exp. 2018 Apr 9(134):57069. doi: 10.3791/57069.
The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.
通过琥珀色终止密码子抑制实现非标准氨基酸（ncAA）的基因掺入是一种在活细胞中直接将人工探针和反应性基团安装到蛋白质上的强大技术。每个ncAA由一对专门的正交抑制性tRNA/氨酰-tRNA合成酶（AARS）掺入，该对被导入宿主生物体。不同ncAA的掺入效率可能有很大差异，在某些情况下并不理想。可以通过操纵AARS或tRNA来改进正交对。然而，使用大型文库和死活筛选方法对tRNA或AARS进行定向进化在哺乳动物细胞中是不可行的。在此，提出了一种简便且稳健的基于荧光的测定方法，用于评估哺乳动物细胞中正交对的效率。该测定方法允许在适度的工作量和合理的时间内筛选数十至数百个AARS/tRNA变体。描述了使用该测定方法生成显著提高吡咯赖氨酸正交系统效率的新tRNA，以及将ncAA应用于G蛋白偶联受体（GPCR）的研究，GPCR是ncAA诱变的具有挑战性的对象。首先，通过在受体的整个细胞外表面系统地掺入光交联ncAA，直接在活细胞中绘制完整受体上不同配体的结合位点。其次，通过将最新一代的ncAA掺入GPCR中，证明了用荧光染料进行超快无催化剂受体标记，这利用了活细胞上的生物正交应变促进的逆狄尔斯-阿尔德环加成反应（SPIEDAC）。由于ncAA通常可以独立于其大小应用于任何蛋白质，因此该方法在许多应用中具有普遍意义。此外，ncAA掺入不需要任何特殊设备，并且在标准生物化学实验室中很容易进行。
