Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes

Genes encoding Arabidopsis subunit homologs of TFIIH kinase module were labelled with coding sequences of GFP and mCherry (green and red fluorescent proteins) using fast-track recombineering. To illustrate the applicability of recombineering for accelerating the isolation and identification of plant protein complexes, proteins associated with CDKD;2-GFP, CYCLIN-H-mCherry and DNA replication-dependent HISTONE H3.1-mCherry complexes were purified on GFP-Trap and RFP-TRAP and analysed by LC-MS/MS mass spectrometry. The results confirmed association of know TFIIH subunit homologs with CDKD;2 and CYCLIN H, and identified subunits of CAF1 (CHROMATIN ASSEMBLY FACTOR 1) and ASF1A/B histone chaperon in complex with HISTONE H3.1.

采用快速重组工程技术，将绿色荧光蛋白（GFP）与红色荧光蛋白（mCherry）的编码序列标记至编码转录因子IIH（TFIIH）激酶模块亚基同源物的拟南芥基因上。为验证重组工程技术在加速植物蛋白质复合物分离与鉴定中的应用潜力，研究人员针对CDKD;2-GFP、细胞周期蛋白H（CYCLIN-H）-mCherry及DNA复制依赖型组蛋白H3.1-mCherry复合物，分别通过GFP-Trap与RFP-TRAP亲和纯化与之结合的蛋白质，并采用液相色谱-串联质谱（LC-MS/MS）进行分析。实验结果证实了已知转录因子IIH亚基同源物与CDKD;2及细胞周期蛋白H的相互作用，并鉴定出与组蛋白H3.1形成复合物的染色质组装因子1（CHROMATIN ASSEMBLY FACTOR 1，简称CAF1）亚基与ASF1A/B组蛋白伴侣蛋白。