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.