Kato Mieko, Chiba Tomoki, Li Min, Hanyu Yoshiro
Signaling Molecules Research Group, Neuroscience Research Institute, National Institutes of Advanced Industrial Science and Technology, Tsukuba, 1-1-1 Higashi, Tsukuba, Japan.
Assay Drug Dev Technol. 2011 Feb;9(1):31-9. doi: 10.1089/adt.2010.0278. Epub 2010 Sep 13.
We have developed a method to measure the amounts of cell surface-expressed membrane proteins with bioluminescence. Dinoflagellate luciferase was expressed on the surface of a mammalian cell as a chimeric fusion protein with a membrane protein of interest. Using a membrane-impermeable substrate to quantify the membrane-displayed luciferase, the expression of the membrane protein on the cell surface was determined. By inclusion of a quenching step for the luminescent activity of luciferase on the cell surface, we were able to monitor the membrane protein expression kinetics by measuring the luminescence recovery from the cell surface after quenching. The reported methods provide a convenient way to monitor the kinetics of expression and transport of membrane proteins to the cell surface. It is applicable to the high-throughput analysis of drugs or drug candidates concerning their effects on membrane protein expression.
我们开发了一种利用生物发光来测量细胞表面表达的膜蛋白数量的方法。将甲藻荧光素酶作为与感兴趣的膜蛋白的嵌合融合蛋白在哺乳动物细胞表面表达。使用一种不能透过细胞膜的底物来定量膜展示的荧光素酶，从而确定膜蛋白在细胞表面的表达。通过对细胞表面荧光素酶的发光活性进行淬灭步骤，我们能够通过测量淬灭后细胞表面的发光恢复来监测膜蛋白表达动力学。所报道的方法提供了一种方便的方式来监测膜蛋白向细胞表面表达和转运的动力学。它适用于对药物或候选药物对膜蛋白表达影响的高通量分析。
