A novel purification method for histidine-tagged proteins containing a thrombin cleavage site

A general procedure for the purification of histidine-tagged proteins has b een developed using immobilized metal-ion affinity chromatography. This two -step purification method can be used for proteins containing a hexahistidi ne tag and a thrombin cleavage site, yielding high amounts of purified prot ein. The advantage of this method is that thrombin is used instead of imida zole in the final purification step. Imidazole can influence NMR experiment s, competition studies, or crystallographic trials, and the presence of imi dazole often results in protein aggregates. Removal of the His-tag results in a form of the protein of interest in which no additional tags are presen t, resembling the native form of the protein, with only three additional am ino acids at the N-terminal side. Our method is compared with a more conven tional method for the purification of the Azotobacter vinelandii NIFL PAS d omain, overexpressed in Escherichia coli. It also proves to be successful f or three different His-tagged proteins, the Klebsiella pneumoniae NTRC prot ein, and the A. vinelandii NIFA and NIFL proteins, and therefore it is a ge neral method for the purification of His-tagged proteins. (C) 2001 Academic Press.