Purification of (His)6EcoRV [recombinant restriction endonuclease EcoRV fused to a (His)6affinity domain] by metal‐chelate affinity chromatography

The chromatographic purification of (His)6EcoRV, a fusion protein consisting of a hexahistidine affinity domain and restriction endonuclease EcoRV produced from recombinant Escherichia coli, led to high product concentrations (> or = 1 mg/ml) in the preparative mode. Increasing the amount of applied crude cell homogenate caused competition with host-specific proteins was achieved by pre-adsorption on to a DEAE anion-exchange sorbent. This, in combination with 0.5-1 M NaCl in the adsorption buffer, assured a purity > 95% and a total protein recovery of approximately 34% in the preparative mode. Contamination of the product with about 2 mol of Ni(11)/mol of (His)6EcoRV was found due to metal-ion transfer to the N-terminal high-affinity binding site at (His)6. Tris(carboxymethyl)ethylenediamine (TED)-Sepharose was employed as an Ni(11) adsorber. One passage of Ni(11)-contaminated protein solutions through the TED-Sepharose column resulted in a decrease in the Ni(11) content in the (His)6EcoRV fractions below the detection limit (approximately 0.02 mg/l) of the atomic-adsorption spectrophotometer.