Recombinant production of proteins is one of the most powerful techniques used in the Life Sciences. The ability to produce and purify a desired recombinant protein in high yield and purity permits a wide range of uses in industrial processes, diagnosis and treatment of diseases, and enabling basic and applied research.
At first glance, protein purification may appear to be a simple, straightforward process. Essentially, the gene of interest is expressed with an affinity tag, such as a polyhistidine tag, or fused to a protein, such as maltose binding protein (MBP). A crude lysate containing the overexpressed gene product is then passed over a specific resin with affinity to bind the tag or fused protein, while other proteins and contaminants are removed during a wash step. Lastly, the target protein is eluted from the affinity resin, resulting in the isolation of highly pure protein in appreciable yields. However, not all proteins are or can be tagged, and different resins, beads or magnetic matrices may need to be used under different buffering conditions (such as varying pH and salt concentrations) to achieve optimal purity of the target protein. Thus, no single solution exists for successful purification of all recombinant proteins. Instead, it is beneficial to have access to a wide range of purification tools and a willingness to explore multiple approaches to better one’s chances for success.
- NEBExpress® Ni Spin Columns Quick Start Protocol (NEB #S1427)
- NEBExpress® Ni Resin Quick Start Protocol (NEB #S1428)
- His-tag removal from protein using TEV Protease
- Affinity Purification and On-column Cleavage (NEB #S6651)
- Small Scale Affinity Chromatography (NEB #E8201)
- Large Scale Affinity Chromatography (NEB #E8201)
- Separating the Protein of Interest from MBP after TEV Protease Cleavage (NEB #E8201)
Over 40 years in protein expression and purification – a historical perspective
This article provides an overview of the advances in protein expression and purification methodology over the past 40 years.
- Competent Cell Brochure
- Protein Expression & Purification Brochure
- Purification Beads, Columns and Resins Brochure
- Competent Cell Product Comparison
- Protein Expression and Purification Selection Chart
- Reuter, W.H., Masuch, T., Ke, N., Lenon, M., Radzinski, M., Van Loi, V., Ren, G., Riggs, P., Antelmann, H., Reichmann, D., Leichert, L.I., Berkmen, M 2019. Utilizing redox-sensitive GFP fusions to detect in vivo redox changes in a genetically engineered prokaryote Redox Biol. 26, PubMedID: 31450103, DOI: 10.1016/j.redox.2019.101280
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