Arabidopsis FPG cDNA, a gift from Scott Kathe and Susan Wallace, University of Vermont, Burlington, VT (31 (link)), was subcloned into pET30b expression vector (Novagen) using XhoI and XbaI sites. Expression was carried out in E. coli BL21 (DE3) dcm− Codon Plus cells (Stratagene) induced during 2 h by adding 1 mM isopropyl-1-thio-β-d -galactopyranoside. His-FPG was purified by affinity chromatography on a Ni2+-Sepharose column (HisTrap HP; GE Healthcare). Protein was eluted with a 60 mM to 1 M gradient of imidazole and analyzed by SDS/PAGE (10%) using broad-range molecular weight standards (Bio-Rad). Protein concentration was determined by the Bradford assay. His-ZDP, MBP-ZDP, and His-ARP were expressed and purified as previously described (23 (link), 25 (link)).
Protocol detail
Purification of Arabidopsis FPG Protein
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Affinity chromatography
Arabidopsis
Assay
Cdna
Cells
Codon
E coli
Galactopyranoside
Imidazole
Protein
Sds page
Sepharose
Vector
Corresponding Organization :
Other organizations : University of Córdoba, Hospital Universitario Reina Sofía, Instituto Maimónides de Investigación Biomédica de Córdoba
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Variable analysis
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- Expression of
Arabidopsis FPG cDNA inE. coli BL21 (DE3)dcm − Codon Plus cells
- Purification of His-FPG by affinity chromatography on a Ni2+-Sepharose column
- Elution of His-FPG with a 60 mM to 1 M gradient of imidazole
- Analysis of His-FPG by SDS/PAGE (10%) using broad-range molecular weight standards
- Determination of His-FPG protein concentration by the Bradford assay
- Expression and purification of His-ZDP, MBP-ZDP, and His-ARP as previously described
- Expression and purification of His-ZDP, MBP-ZDP, and His-ARP
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