Common protein tags explained- Strep
Based on the natural affinity between biotin and streptavidin, Strep-tag is one of the most specific-binding speciality tags on the market. Its high specificity positions it as an alternative or supplement to His-trap, which is based on a non-specific binding of His to metal ions in resin. This blog post outlines the features of Strep-tag and how it compares to TriAltus’ CL7.Strep-tag
Strep-tag II (8 amino acids) and Twin Strep-tag (28 amino acids) are two affinity tag options for expressing with the protein of interest (IBA Life Sciences). These synthetic peptides exhibit affinity to streptavidin variants used as resin ligands. Strep-tag and Twin Strep-tag have binding affinities to resins Strep-Tactin and Strep-Tactin XT in the nM to pM range.
Twin Strep-Tag’s nearly covalent bond to Strep-Tactin XT resin allows for no off-target binding, a benefit over His-tag. Strep-Tactin XT resin allows for higher binding capacity than regular Strep-Tactin because of its higher binding affinity to the Twin Strep-tag. Among the Strep-Tactin XT resin varieties, Strep-Tactin XT has a reported 4.1 mg protein/ml resin binding capacity while the high capacity resin reports about 16 mg/ml capacity. The resin is regenerable using NaOH 3-5 times.
Strep-tag is also small enough to not be cleaved from the final protein product. This can be seen as a benefit or a liability, depending on the application for the purified protein.
However, the Strep-tag system has several limitations. Like many other specialized tags on the market, Strep-tag has a high sensitivity to high salt loading buffers. This limits the purity that can be achieved in some cases, and requires more chromatography steps to result in an ultra-pure product. High salt sensitivity can also be a roadblock to purifying difficult classes of proteins such as DNA-binding proteins that are best purified when all nucleic acids are removed in the early washes.
- Strong and highly specific binding affinity
- Low resin binding capacity
- Sensitive to high salt buffers
- Tag not generally cleaved
Comparison to CL7
The high, salt-independent binding affinity between CL7 and Im7 allows for earlier removal of impurities. CL7’s salt tolerance allows even loading in high salt, which is advantageous because some contaminants, once bound to the column, may not be removed even with high salt washes later. This results in a higher purity in only one chromatography step. CL7 also confers some added solubility which helps when purifying proteins that usually aggregate in inclusion bodies and those that are multi-subunit. Additionally, the Im7 resin has a very high binding capacity of about 40 mg/ml and is reusable up to 100x when regenerated using Guanidine.