Facile and scalable expression and purification of transcription factor IIH (TFIIH) core complex
| Autor: | Biljana Petrovic-Stojanovska, Reyes Sanles-Falagan, Malcolm F. White |
|---|---|
| Přispěvatelé: | BBSRC, University of St Andrews. School of Biology, University of St Andrews. Biomedical Sciences Research Complex |
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
2A-sequences Sf9 High FiveTM Gene Expression MultiBacTM QH426 Genetics Spodoptera 01 natural sciences 03 medical and health sciences chemistry.chemical_compound Plasmid SDG 3 - Good Health and Well-being Transcription (biology) Size exclusion chromatography 010608 biotechnology RNA polymerase TFIIH Core Sf9 Cells Animals Humans QH426 030304 developmental biology 0303 health sciences Expression vector TEV cleaving Immobilised metal ion affinity chromatography DAS QR Microbiology Heparin chromatography Recombinant Proteins Cell biology QR Transcription Factor TFIIH chemistry Transcription factor II H DNA Nucleotide excision repair Biotechnology |
| Zdroj: | Protein expression and purification. 174 |
| ISSN: | 1096-0279 |
| Popis: | This work was supported by the BBSRC [grant number BB/J01446X/1] and [grant number BB/R015570/1] Transcription factor IIH (TFIIH) plays essential roles in both the initiation of RNA Polymerase II-mediated transcription and the Nucleotide Excision Repair (NER) pathway in eukaryotes. In NER, the 7-subunit TFIIH Core sub-complex is responsible for the opening and extension of the DNA bubble created at the lesion site, utilizing the molecular motors XPB and XPD. Mutations in Core subunits are associated with a series of severe autosomal recessive disorders characterised by symptoms such as mild-to-extreme photosensitivity, premature ageing, physical and neurological anomalies, and in some cases an increased susceptibility to cancer. Although TFIIH Core has been successfully obtained in the past, the process has always remained challenging and laborious, involving many steps that severely hindered the amount of pure, active complex obtained. This has limited biochemical and functional studies of the NER process. Here we describe improved and simplified processes for the cloning, expression and purification of the 7-subunit TFIIH Core sub-complex. The combined use of auto-cleavable 2A-like sequences derived from the Foot-and-Mouth Disease Virus (FMDV) and the MultiBac™ cloning system, a powerful baculoviral expression vector specifically conceived for the obtaining of multi-subunit eukaryotic complexes, allowed us to obtain a single, 7-gene plasmid in a short time using regular restriction cloning strategies. Additionally, expression of the construct in High Five™ insect cells paired with a simple 5-step purification protocol allowed the extraction of a pure, active TFIIH Core sub-complex in milligram quantities. Postprint |
| Databáze: | OpenAIRE |
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