Olejnik Jerzy, Gite Sadanand, Mamaev Sergey, Rothschild Kenneth J
AmberGen, 1106 Commonwealth Avenue, Boston, MA 02215, USA.
Methods. 2005 Jul;36(3):252-60. doi: 10.1016/j.ymeth.2005.04.003.
Methodology based on tRNA mediated protein engineering is described for the introduction of fluorophores and other labels at the N-terminus of proteins produced in cell-free translation systems. One method for low-level (trace) N-terminal labeling is based on the use of an Escherichia coli initiator tRNA(fMet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, the protein translational machinery incorporates the fluorophore BODIPY-FL and the affinity tag biotin at an N-terminal end of the nascent protein. A second method for higher N-terminal labeling uses a chemically aminoacylated amber initiator suppressor tRNA and a DNA template which contains a complementary amber (UAG) codon instead of the normal initiation (AUG) codon. This more versatile approach is demonstrated using a variety of N-terminal markers including fluorescein, biotin, PC-biotin, and a novel dual marker conjugate (Biotin/BODIPY-FL).
本文描述了一种基于tRNA介导的蛋白质工程方法，用于在无细胞翻译系统中产生的蛋白质的N端引入荧光团和其他标记。一种用于低水平（微量）N端标记的方法是基于使用一种用α-氨基修饰的甲硫氨酸错误氨酰化的大肠杆菌起始tRNA（fMet）。除了正常的甲酰基外，蛋白质翻译机制还在新生蛋白质的N端掺入荧光团BODIPY-FL和亲和标签生物素。另一种用于更高水平N端标记的方法是使用化学氨酰化的琥珀酸起始抑制tRNA和一个DNA模板，该模板包含一个互补的琥珀酸（UAG）密码子而不是正常的起始（AUG）密码子。使用包括荧光素、生物素、PC-生物素和一种新型双标记共轭物（生物素/BODIPY-FL）在内的多种N端标记物证明了这种更通用的方法。
