Expression of FLAG fusion proteins in insect cells: Application to the multi-subunit transcription/DNA repair factor TFIIH

被引：13

作者：

Jawhari, A ^{[1
]}

Uhring, M ^{[1
]}

Crucifix, C ^{[1
]}

Fribourg, S ^{[1
]}

Schultz, P ^{[1
]}

Poterszman, A ^{[1
]}

Egly, JM ^{[1
]}

Moras, D ^{[1
]}

机构：

[1] CU Strasbourg, Inst Genet & Biol Cellulaire & Mol, CNRS, INSERM,ULP,Coll France, F-67404 Illkirch Graffenstaden, France

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2002年 / 24卷 / 03期

关键词：

D O I：

10.1006/prep.2001.1597

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The multi-subunit transcription/DNA repair factor TFIIH was used as a model system to show that the expression of FLAG fusion proteins in insect cells constitutes a versatile tool for both structural and functional investigations. In the present study, we have constructed recombinant baculoviruses expressing the four core TFIIH subunits fused at their N-terminus to the FLAG peptide. Using these recombinant viruses we have established protocols based on anti-FLAG immunoaffinity chromatography that allow the systematic analysis of pairwise interaction within multiprotein complexes and have developed a double tag strategy (FLAG and hexahistidine tags) for the identification and purification of stable TFIIH subcomplexes. A simple purification procedure was developed that leads to the isolation of recombinant TFIIH containing the fall set of subunits. The purified recombinant TFIIH was shown to be active in a transcription assay and to be structurally homologous to the endogenous complex by electron microscopy and image analysis. (C) 2002 Elsevier Science (USA).

引用

页码：513 / 523

页数：11

共 18 条

[1] Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH [J].