CF IB was expressed and purified as described previously (Casañal et al. 2017 (link)). A DNA sequence encoding full-length Pab1p from S. cerevisiae was codon-optimized for E. coli (GenScript) and cloned into a pGEX-6P-1 vector containing an N-terminal GST-tag with an HRV-3C protease cleavage site. Protein expression in BL21 DE3 was induced using 0.5 mM IPGT, cells harvested by centrifugation, and lysed by sonication. The clear supernatant was applied to a 5-mL GSTrap 4B column (Cytiva) followed by an overnight incubation at 4°C with a 1:50 molar ratio (protein:enzyme) of HRV-3C protease to remove the GST-tag. The peak fractions were pooled and applied to a Superdex 200 increase 10/300 column (Cytiva), analysed by SDS-PAGE, concentrated to 5 mg/mL, and flash-frozen in liquid nitrogen for storage at −80°C.
Gstrap 4b column
Manufactured by Cytiva
The GSTrap 4B column is a pre-packed affinity chromatography column designed for the purification of recombinant proteins fused with glutathione S-transferase (GST) tags. The column matrix is made of agarose beads that have been coupled with glutathione, which binds to the GST tag on the target protein. This allows for the selective capture and purification of the GST-tagged protein from complex samples.
Automatically generated - may contain errors
2 protocols using gstrap 4b column
1
Purification of Pab1p from S. cerevisiae
2
Purification of Recombinant Prion Proteins
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Soluble forms of huPrP-GST, PrP125-GST, PrP174-GST, and PrP180-GST protein were collected and filtered by 0.2 μm membrane before loading into GSTrap 4B column (Cytiva) using ÄKTA protein purification system (Cytiva). Then, the contaminants were removed by binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4); following that, the target proteins were eluted with elution buffer (50 mM Tris–HCl, 10 mM reduced glutathione, pH 8.0) [21 (link)].
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