Human APT1 and APT2 wildtype and mutant proteins were expressed and purified as described elsewhere 21 (link) with minor modifications. Briefly, BL21 [DE3] (Novagen 70235-3) harboring the appropriate plasmids were grown at 37°C to OD600 = 0.6. Protein expression was induced by the addition of IPTG to 0.8 mM and cultures were grown for an additional 18 h at 16°C. Cells were lysed by sonication in ice-cold H Buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl), and cellular debris was pelleted by centrifugation at 15,000 × g for 30 min at 4°C. The supernatant was loaded on a HisTrap HP (GE LifeSciences 17524802), and protein was eluted in H Buffer containing 250 mM imidazole. Where applicable, the N-terminal His6-tag was removed by the addition of His6-rTEV protease (Life Technologies) and dialyzed in H Buffer for 36 h at 4°C. Cleaved protein was harvested, and DTT and EDTA were added to concentrations of 5 mM and 1 mM, respectively.
His6 rtev protease
Manufactured by Thermo Fisher Scientific
His6-rTEV protease is a recombinant fusion protein that includes a 6-histidine tag and a Tobacco Etch Virus (TEV) protease. It is designed to cleave the TEV recognition sequence from target proteins.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using his6 rtev protease
1
Purification of Human APT1 and APT2 Proteins
2
Purification of Human APT1 and APT2 Proteins
3
Purification of Human APT1 and APT2
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Human APT1 and APT2 wildtype and mutant proteins were expressed and purified as described elsewhere (Devedjiev et al, 2000) with minor modifications. Briefly, BL21
[DE3] (Novagen 70235-3) harboring the appropriate plasmids were grown at 37°C to OD600 = 0.6. Protein expression was induced by the addition of IPTG to 0.8 mM and cultures were grown for an additional 18 h at 16°C. Cells were lysed by sonication in ice-cold H Buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl), and cellular debris was pelleted by centrifugation at 15,000 × g for 30 min at 4°C. The supernatant was loaded on a HisTrap HP (GE LifeSciences 17524802), and protein was eluted in H Buffer containing 250 mM imidazole. Where applicable, the N-terminal His6-tag was removed by the addition of His6-rTEV protease (Life Technologies) and dialyzed in H Buffer for 36 h at 4°C. Cleaved protein was harvested, and DTT and EDTA were added to concentrations of 5 mM and 1 mM, respectively.
[DE3] (Novagen 70235-3) harboring the appropriate plasmids were grown at 37°C to OD600 = 0.6. Protein expression was induced by the addition of IPTG to 0.8 mM and cultures were grown for an additional 18 h at 16°C. Cells were lysed by sonication in ice-cold H Buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl), and cellular debris was pelleted by centrifugation at 15,000 × g for 30 min at 4°C. The supernatant was loaded on a HisTrap HP (GE LifeSciences 17524802), and protein was eluted in H Buffer containing 250 mM imidazole. Where applicable, the N-terminal His6-tag was removed by the addition of His6-rTEV protease (Life Technologies) and dialyzed in H Buffer for 36 h at 4°C. Cleaved protein was harvested, and DTT and EDTA were added to concentrations of 5 mM and 1 mM, respectively.
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