Two-step metal affinity purification of double-tagged (NusA–His6) fusion proteins
- 1 August 2006
- journal article
- Published by Springer Nature in Nature Protocols
- Vol. 1 (3) , 1538-1543
- https://doi.org/10.1038/nprot.2006.289
Abstract
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.Keywords
This publication has 13 references indexed in Scilit:
- A family of E. coliexpression vectors for laboratory scale and high throughput soluble protein productionBMC Biotechnology, 2006
- Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectorsMicrobial Cell Factories, 2005
- Gateway vectors for the production of combinatorially‐tagged His6‐MBP fusion proteins in the cytoplasm and periplasm of Escherichia coliProtein Science, 2005
- Making the most of affinity tagsTrends in Biotechnology, 2005
- The solubility and stability of recombinant proteins are increased by their fusion to NusABiochemical and Biophysical Research Communications, 2004
- A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoproteinProtein Engineering, Design and Selection, 2001
- New fusion protein systems designed to give soluble expression inEscherichia coliBiotechnology & Bioengineering, 1999
- High-Level Expression of Soluble Protein inEscherichia coliUsing a His6-Tag and Maltose-Binding-Protein Double-Affinity Fusion SystemProtein Expression and Purification, 1997
- Conformational stability of pGEX‐expressed Schistosoma japonicum glutathione S‐transferase: A detoxification enzyme and fusion‐protein affinity tagProtein Science, 1997
- Multiple affinity domains for the detection, purification and immobilization of recombinant proteinsJournal of Molecular Recognition, 1996