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
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
收藏
页码:513 / 523
页数:11
相关论文
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].
Coin, F ;
Marinoni, JC ;
Rodolfo, C ;
Fribourg, S ;
Pedrini, AM ;
Egly, JM .
NATURE GENETICS, 1998, 20 (02) :184-188
[2]   Mutations in XPB and XPD helicases found in xeroderma pigmentosum patients impair the transcription function of TFIIH [J].
Coin, F ;
Bergmann, E ;
Tremeau-Bravard, A ;
Egly, JM .
EMBO JOURNAL, 1999, 18 (05) :1357-1366
[3]   Reconstitution of recombinant TFIIH that can mediate activator-dependent transcription [J].
Fukuda, A ;
Yamauchi, J ;
Wu, SY ;
Chiang, CM ;
Muramatsu, M ;
Hisatake, K .
GENES TO CELLS, 2001, 6 (08) :707-719
[4]  
GERARD M, 1991, J BIOL CHEM, V266, P20940
[5]   TFIIH: A key component in multiple DNA transactions [J].
Hoeijmakers, JHJ ;
Egly, JM ;
Vermeulen, W .
CURRENT OPINION IN GENETICS & DEVELOPMENT, 1996, 6 (01) :26-33
[6]   Interactions involving the human RNA polymerase II transcription/nucleotide excision repair complex TFIIH, the nucleotide excision repair protein XPG, and Cockayne syndrome group B (CSB) protein [J].
Iyer, N ;
Reagan, MS ;
Wu, KJ ;
Canagarajah, B ;
Friedberg, EC .
BIOCHEMISTRY, 1996, 35 (07) :2157-2167
[7]   Epitope tagging [J].
Jarvik, JW ;
Telmer, CA .
ANNUAL REVIEW OF GENETICS, 1998, 32 :601-618
[8]   Immunoaffinity purification and functional characterization of human transcription factor IIH and RNA polymerase II from clonal cell lines that conditionally express epitope-tagged subunits of the multiprotein complexes [J].
Kershnar, E ;
Wu, SY ;
Chiang, CM .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (51) :34444-34453
[9]   Baculoviral transfer vectors for expression of FLAG(R) fusion proteins in insect cells [J].
Koh, SS ;
Hengartner, CJ ;
Young, RA .
BIOTECHNIQUES, 1997, 23 (04) :622-&
[10]   Immunoaffinity purification of the human multisubunit transcription factor IIH [J].
LeRoy, G ;
Drapkin, R ;
Weis, L ;
Reinberg, D .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (12) :7134-7140