Protein tags and modifiers are short peptide sequences cloned in frame with protein coding sequences that change the protein's behavior. Affinity tags are generally appended to the N- or C-terminus of proteins to enable ready purification of the protein from cells.
Generally, cells expressing the protein fused to an affinity tag are pelleted, lysed, and applied to a column, resin or other solid support that displays a ligand to the affinity tags. The solid support is washed several times with buffer to eliminate unbound proteins. Then the protein of interest is eluted from the solid support via a buffer that causes the affinity tag to dissociate from the ligand resulting in a purified protein.
There are several different affinity tags available for purification. Each has benefits and advantages depending on your protein of interest and the cell from which you are purifying it.
Cleavage sites are specific peptide sequences, or more often, peptide motifs at which site-specific proteases with cleave or cut the protein. Cleavage sites can be used, for example, to cleave off an affinity tag thereby restoring the natural protein sequence or to inactivate a protein.
Degradation tags are short peptide sequences that mark a protein for degradation by the cell's protein recycling machinery. In doing so, the degradation tag effectively decreases the protein half-life or the typical length of time that a protein, once translated, will exist in the cell. (Formally, the half-life is the interval time it takes for the level of the protein to decay to half its initial value.) In practice, adding a degradation tag will also tend to decrease the concentration of the protein in the cell.
Many of the degradation tags available via the Registry are "ssRA tags" which function in E. coli. In the natural system, ssRA tagging occurs when a ribosome gets stuck on a broken ("truncated") mRNA. Without a normal termination codon, the ribosome cannot detach from the defective mRNA. A special type of RNA known as ssRA ("small stable RNA A") or tmRNA ("transfer-messenger RNA") rescues the ribosome by adding an eleven codon degradation tag followed by a stop codon. This allows the ribosome to break free and continue functioning. The tagged, incomplete protein then gets degraded by the proteases ClpXP or ClpAP.
Although the originally identified ssRA/tmRNA tag had the sequence
AANDENYALAA Keiler, additional designed degradation tags that vary in the final three amino acids (
AAV, ASV, LVA, LAA) have since been found that result in different protein half-lives Andersen.
Linkers are short peptide sequences that occur between protein domains. Linkers are often composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers are used when it is necessary to ensure that two adjacent domains do not sterically interfere with one another.
Certain protein tags can direct a protein to a particular physical location in the cell, such as the nucleus, the membrane, the periplasm, secretion outside of the cell or elsewhere. The localization tags are useful for achieving spatial segregation of proteins.