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Abstract
This protocol describes the production of GST-Cbx7 fusion proteins from E. coli, originally developed in the recent publication (Zhen et al., 2016). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells. The fusion protein production was induced by isopropyl-beta-D-thiogalactopyranoside and they were purified by Glutathione Sepharose 4B. This protocol can be adapted for the purification of other proteins.
Highlights
Materials and Reagents3. Bio-Rad Chromatography Column (2.5 x 10 cm Econo-Column) (Bio-Rad Laboratories, catalog number: 7311550)
[Background] Polycomb group (PcG) proteins regulate gene expression by modulating higher order chromatin structures (Kerppola, 2009; Simon and Kingston, 2013)
PcG proteins are generally found in two major complexes, Polycomb repressive complex (PRC) 1 and 2 (Kerppola, 2009; Simon and Kingston, 2013)
Summary
3. Bio-Rad Chromatography Column (2.5 x 10 cm Econo-Column) (Bio-Rad Laboratories, catalog number: 7311550). Isopropyl-1-thio-β-D-galactopyranoside (IPTG) (Omega Bio-tek, catalog number: AC121) 8. Glutathione Sepharose 4B (GE Healthcare, catalog number: 17075601) 9. PierceTM Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23200) 11. Coomassie Blue (Bio-Rad Laboratories, catalog number: 1610786) 12. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 93482) 17. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340) 18. L-glutathione reduced (Sigma-Aldrich, catalog number: G4251) 19. Vibra-CellTM sonicator (Sonics & Materials, model: VCX 130) with standard probe (1/4”; 6 mm), length 4.5” (113 mm), Titanium alloy Ti-6Al-4V, Autoclavable (Sonics & Materials, catalog number: 630-0435) 6. S-3200-2 GyroMixer (BioExpress, model: GeneMate GyroMixer, catalog number: S-3200-2)
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