Recombinant protein purification definition

The 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.

When a protein is fused to a peptide or protein tag, such as polyhistidine (His), glutathione-S-transferase (GST), maltose binding protein (MBP), or Strep-tag II, the properties of the tag can be exploited for purification purposes. Affinity chromatography methods have been developed for each of the commonly used tags, and there is a good chance of a successful purification of a tagged protein in a single step.

Protein fusion tags are indispensible tools used to improve purification yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts.

Application

Useful applications of affinity-tag purification schemes include:

1. Developing and producing proteins for therapeutic applications
2. Developing vaccines
3. Generating sufficient protein for crystallization and structural analysis
4. Identifying protein binding partners to elucidate functional pathways
5. Determining cellular localization, identifying posttranslational modifications, and biochemically characterizing proteins
6. Characterizing protein properties via site-directed mutagenesis

Tag

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).

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 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 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.