A novel purification method for histidine-tagged proteins containing a thrombin cleavage site.
A general procedure for the purification of histidine-tagged proteins has been developed using immobilized metal-ion affinity chromatography. This two-step purification method can be used for proteins containing a hexahistidine tag and a thrombin cleavage site, yielding high amounts of purified protein. The advantage of this method is that thrombin is used instead of imidazole in the final purification step. Imidazole can influence NMR experiments, competition studies, or crystallographic trials, and the presence of imidazole 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 present, resembling the native form of the protein, with only three additional amino acids at the N-terminal side. Our method is compared with a more conventional method for the purification of the Azotobacter vinelandii NIFL PAS domain, overexpressed in Escherichia coli. It also proves to be successful for three different His-tagged proteins, the Klebsiella pneumoniae NTRC protein, and the A. vinelandii NIFA and NIFL proteins, and therefore it is a general method for the purification of His-tagged proteins.
Hefti
M H
MH
Laboratory of Biochemistry, Wageningen University, Dreyenlaan 3, 6703 HA Wageningen, The Netherlands.
Van Vugt-Van der Toorn
C J
CJ
Dixon
R
R
Vervoort
J
J
D016948:Azotobacter vinelandii; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006639:Histidine / Q000737:chemistry; D007093:Imidazoles; D007711:Klebsiella pneumoniae; D051922:PII Nitrogen Regulatory Proteins; D013917:Thrombin; D015534:Trans-Activators; D014157:Transcription Factors
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D007093:Imidazoles; D051922:PII Nitrogen Regulatory Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli; C084296:nifL protein, Bacteria; D006639:Histidine; D013917:Thrombin