Two-step metal affinity purification of double-tagged (NusA-His6) fusion proteins.
The present purification protocol applies to target proteins that are fused to a double tag, such as NusA-His6, through a linker that includes a protease-recognition sequence. It involves two steps of immobilized metal ion affinity chromatography (IMAC). NusA stabilizes the passenger protein during translation, whereas the His-tag enables affinity purification of the fusion. The eluate resulting from the first IMAC is buffer-exchanged to remove the imidazole and to achieve optimal conditions for the enzymatic cleavage performed by a His-tagged recombinant protease. The digested sample is loaded directly for a second IMAC step and the target protein is selectively recovered in the flow-through. The resin binds residual non-digested fusion protein, double-tagged moiety, protease and any contaminant that bound the affinity resin and was eluted from the first IMAC. The purity of the target protein usually makes a further purification step unnecessary for most of the lab applications. It takes less than 5 hours to purify the protein from a 5 g pellet.
de Marco
Ario
A
IFOM-IEO Campus for Oncogenomics, via Adamello 16, I-20139, Milano, Italy. ario.demarco@ifom-ieo-campus.it
D019747:Aldose-Ketose Isomerases / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D029968:Escherichia coli Proteins / Q000378:metabolism; D008670:Metals; D010445:Peptide Elongation Factors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D029968:Escherichia coli Proteins; D008670:Metals; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D019747:Aldose-Ketose Isomerases; C079656:Phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxiamide isomerase