Low protein yield is a common issue in protein purification, especially when it comes to isolating large or complex structures. In lieu of using denaturants to elute protein from an affinity column, protease elution can be a welcome alternative to give tag-free protein. In order to ensure the best possible results from proteolytic cleavage, there are three major factors to keep in mind as you prepare your purification.
1. Minimize interference from contaminants
Contaminants in your protein sample may cause blockage. They could bind the target protein and prevent it from binding to the column. For example, leftover DNA fragments in cell lysate are attracted to DNA-binding proteins.
To reduce contamination, load the cell lysate in a high salt buffer. The loading stage is the best time to use a high salt loading buffer because impurities are easier to remove than in later wash stages. With minimal interference the protease can work unhindered and reach the target protein to remove it from the column.
2. Check the pH of solution
Proteases have an optimal pH range in which they will cleave best. SUMO works best from pH 5.5-9.5 and PreScission from pH 7-8. Every affinity purification system also has its own ideal pH range; Im7 stays crosslinked to resin beads from pH 3-10 but the CL7 tag will only bind Im7 from pH 4.2-10. Regardless of the tag system or proteases used, you will achieve the best results when the proteases operate in the range of the system as a whole. If matching the pH ranges is not possible due to specific limitations, more protease should be added or given more time to digest.
3. Be wary of steric hindrance
Some proteins are large or complex enough that proteases may not be able to reach the cleavage site as easily. For samples of large proteins with very low yield, extra protease can be loaded to give it extra digestive power. The protease can be removed using another column step at the end if desired. During the cloning/design stage, additional spacer or linker residues may be added between the large target protein and the tag to allow more access to the protease. Another option for troubleshooting is to elute the protein using Guanidine and run it on a gel. The gel would show if the protein was cleaved and got stuck to the column or if it was never cleaved in the first place.
Proteases are a powerful tool for achieving high-purity, tag-free protein. Optimizing the conditions in which they work will increase the chances of achieving a spectacular protein sample.