Recombinant tagged purification is a widely used method for purifying proteins of interest based on well-developed recombinant DNA and protein expression technologies. Theoretically, all proteins can be purified using this method regardless of the solubility of the proteins produced in expression host cells. This section provides an overview of tagged recombinant protein affinity purification and discusses specific tagged purifications, such as polyhistidine (His) (Profinity™ IMAC resin), glutathione-S-transferase (GST), (Profinia™ GST resin), and Profinity eXact™ resin. General considerations and trouble shooting tips are discussed for each of these methods. Learn about various methods for protein purification by affinity chromatography using media based on protein A/G and other affinities or activated supports.
Related Topics: Affinity Chromatography, Protein A/G Affinity, and Activated Supports.
Understanding cellular processes requires knowledge of the structure, function, posttranslational modifications, and interactions of proteins. Until recently, progress in understanding proteins had been slowed by the difficulty in purifying proteins. The advent of easy-to-use and high-throughput methods for protein purification should accelerate proteomics research. Useful applications of affinity-tag purification schemes include:
The two most commonly used tags are the polyhistidine tag generally consisting of six 10 histidine residues and GST.
Proteins tagged with histidine bind strongly with metal ions, such as Ni2+, Co2+, Cu2+, and Zn2+. They and are purified by binding to a metal ion immobilized on a support resin by the IMAC method (immobilized metal ion affinity chromatography). Either end of the recombinant protein can be tagged with histidine. Optimal placement is empirically determined and can vary from protein to protein.
For GST tags, the purification principle is based on the binding of GST to gluthathione immobilized on the support resin. After sample impurities are washed from the resin, the bound GST-tagged protein is eluted by reduced glutathione. The GST protein has a molecular weight of 26 kD and is most often fused to the target protein at the N-terminus, though it can work well with C-terminal fusions. The GST protein is a dimer in solution and, thus, the fusion protein dimerizes as well. GST tags can be used to increase solubility of the recombinant protein.
Bio-Rad offers a unique fusion tag purification system. The Profinity eXact fusion tag purification system is an E.coli-based expression and purification system that generates a tag-free, highly purified protein containing its native N-terminal amino acid sequence in a single step without the addition of protease.
This tag system utilizes a modified form of the subtilisin protease, which is immobilized onto a chromatographic support.
The tag is the prodomain (prosignal sequence) of the subtilisin protease, a 75 amino acid sequence that is fused to the N-terminus of the target protein of interest. The mature and prodomain subtilisin protease sequences have been co-engineered to produce a highly specific, high-affinity interaction between the binding partners (KD <100 pm).
Application of the elution buffer triggers the subtilisin processing activity, which quickly and precisely cleaves the tag from the fusion protein and releases the purified protein target. The tag remains tightly bound to the resin and contains only its native amino acid sequence. The structure and activity of the native protein is maintained and the need for cleavage enzymes and purification resin for post-cleavage enzyme removal is eliminated.
Purification and on-column tag cleavage using the Profinity eXact fusion-tag system. During sample application, immobilized subtilisin protease (ligand) recognizes and binds the affinity-tagged protein. Washing the column removes unbound contaminants. Application of fluoride-containing elution buffer triggers subtilisin to quickly and precisely cleave the tag from the fusion protein after the FKAL cleavage recognition sequence. The tag remains tightly bound to the resin, and a highly purified protein with only its native amino acid sequence is released.
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