Facile and scalable expression and purification of transcription factor IIH (TFIIH) core complex

Reyes Sanles-Falagan, Biljana Petrovic-Stojanovska, Malcolm F. White*

*Corresponding author for this work

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Abstract

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.
Original languageEnglish
Article number105660
JournalProtein Expression and Purification
Volume174
Early online date27 May 2020
DOIs
Publication statusPublished - Oct 2020

Keywords

  • TFIIH Core
  • 2A-sequences
  • MultiBacTM
  • Sf9 High FiveTM
  • Immobilised metal ion affinity chromatography
  • Heparin chromatography
  • TEV cleaving
  • Size exclusion chromatography

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