Expression of FLAG fusion proteins in insect cells: application to the multi-subunit transcription/DNA repair factor TFIIH.
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 full 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.
Jawhari
Anass
A
Institut de Génétique et de Biologie Cellulaire et Moléculaire, 67404 Illkirch Cedex, C.U. de Strasbourg, France.
Uhring
Muriel
M
Crucifix
Corinne
C
Fribourg
Sébastien
S
Schultz
Patrick
P
Poterszman
Arnaud
A
Egly
Jean Marc
JM
Moras
Dino
D
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002846:Chromatography, Affinity; D000939:Epitopes; D008958:Models, Molecular; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D018411:Spodoptera / Q000235:genetics; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D019473:Transcription Factors, TFII
D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; C085706:FLAG peptide