How can the yield of purified HAT- or 6xHis-tagged fusion protein be increased?
How can the yield of purified HAT- or 6xHis-tagged fusion protein be increased?
There are different factors that can affect and increase the yield of purified protein:
-Temperature
Always use cold (4–8°C) solutions during extraction, and centrifuge at cool temperatures as well. Performing extractions and purifications at 4°C may reduce protein degradation.
-Use of protease inhibitors
If proteolysis is suspected to be occurring during the extraction process, consider using extraction buffers containing Takara Bio's ProteoGuard EDTA-Free Protease Inhibitor Cocktail (Cat. # 635672). EDTA should not be used as a metalloprotease inhibitor, due to its capacity to chelate metals. If EDTA is used, it must be removed from the sample by gel filtration (PD-10 column, GE Healthcare, Cat. # 17-0851-01) or dialysis, just prior to starting TALON purification.
-Loading/equilibration buffer pH
If yields are still low, the pH of the loading/equilibration buffer can be raised to 8, which increases protein binding to TALON resin.
-Batch purification
Increasing the binding time of the his-tagged protein by employing a batch method of purification can also maximize yield. It is possible to allow the lysate sample to mix with the resin on a rocker or shaker with gentle agitation as long as overnight at 4°C to ensure that all his-tagged proteins are bound to the resin.
-His-tag inaccessibility
If optimizing any of the above factors does not increase yield, try to determine whether or not the protein is passing through the resin without binding by performing a Western blot analysis of the cell extract using a 6xhis monoclonal antibody.
The Western blot should contain samples of the cell extract prior to column loading, the column flowthrough, the wash fractions, and the eluate. Unbound protein will be present in the flowthrough if it does not bind to the column. Problems with protein binding can indicate improper protein folding which results in the inaccessibility of the his-tag.
To test for his-tag inaccessibility, add denaturing reagents such as guanidine-HCl or urea to a few ml of the supernatant and then add this denatured supernatant to a 50 µl aliquot of the resin. Load the column with this material. Successful protein binding followed by elution of his-tagged protein confirms the inaccessibility of the tag—and suggests that the target protein yield will be improved if this protein is purified under denaturing conditions.
-Expression of multiple polyhistidine-containing proteins
Another challenge in purifying proteins from eukaryotic cells is that such cells tend to express a number of polyhistidine-containing proteins. Therefore, we suggest a washing step with 10–20 mM imidazole prior to elution in order to increase the purity of the eluted protein.
-Protein size and charge
It is important to remember that the ability of a protein to bind the resin also depends on its size and overall charge.