Shamonova M A, Koriagina M S, Shliapina V L, Dontsova O A, Rubtsova M P
Faculty of Chemistry, Moscow State University, Moscow, Russia.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Dokl Biochem Biophys. 2025 Sep 21. doi: 10.1134/S1607672925600514.
The functioning of proteins in a cell cannot be studied without analyzing their content in the cell. The most commonly used method of analysis, Western blotting, cannot always be used due to the impossibility of obtaining antibodies that specifically recognize the protein of interest. At the same time, Western blotting is a semi-quantitative method of analysis and does not allow determining the exact content of protein in the cell. In this work, we used the protein complementation method for the qualitative and quantitative assessment of the hTERP protein content in the cell. Using genome editing, a HiBiT epitope was introduced to the C-terminus of the hTERP protein, the complementation of which with LgBiT restores active luciferase. As a result, we determined the hTERP protein content in the HEK293T cell line.
如果不分析蛋白质在细胞中的含量,就无法研究其在细胞中的功能。最常用的分析方法——蛋白质免疫印迹法,由于无法获得能特异性识别目标蛋白质的抗体,并不总是适用。同时,蛋白质免疫印迹法是一种半定量分析方法,无法确定细胞中蛋白质的确切含量。在这项工作中,我们使用蛋白质互补法对细胞中hTERP蛋白的含量进行定性和定量评估。通过基因组编辑,将一个HiBiT表位引入到hTERP蛋白的C端,它与LgBiT互补可恢复活性荧光素酶。结果,我们测定了HEK293T细胞系中hTERP蛋白的含量。