一种基于突变萤火虫荧光素酶功能互补的蛋白质-蛋白质相互作用检测方法FlimPIA

A Protein-Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases.

作者信息

Ohmuro-Matsuyama Yuki, Ueda Hiroshi

机构信息

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.

Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan.

出版信息

Methods Mol Biol. 2016;1461:131-42. doi: 10.1007/978-1-4939-3813-1_10.

DOI:10.1007/978-1-4939-3813-1_10

PMID:27424900

Abstract

There is a significant focus on detecting and assaying protein-protein interactions (PPIs) in biology and biotechnology. Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI by splitting the enzyme-coding or fluorescent protein-coding polypeptide, as well as Förster resonance energy transfer (FRET). Here, we describe a novel PPI assay FlimPIA (firefly luminescent intermediate-based protein-protein interaction assay) by a unique approach of splitting the two major catalytic steps (half reactions) of firefly luciferase (FLuc).

摘要

在生物学和生物技术领域，人们非常关注蛋白质 - 蛋白质相互作用（PPI）的检测和分析。蛋白质片段互补分析（PCA）是通过拆分酶编码或荧光蛋白编码多肽来检测PPI的最广泛使用的方法之一，此外还有福斯特共振能量转移（FRET）。在此，我们描述了一种新型的PPI分析方法FlimPIA（基于萤火虫发光中间体的蛋白质 - 蛋白质相互作用分析），该方法采用了一种独特的方法，即拆分萤火虫荧光素酶（FLuc）的两个主要催化步骤（半反应）。