I am refolding a hydrophobic protein, a fusion tag (size: ~12 kDa; PI: 5.8) from solubilized IB in 8M Urea. The refolding buffer consists of 100mM Tris-HCl, 100mM EDTA, 2M Arginine, 2M Proline, 2% Glycerol pH 8.0. The refolding is done by slow dilution and incubated at 4 oC overnight. The Urea concentration is 1M at the end of dilution. The hydrophobic protein does not have disulfide linkages. An hydrolase enzyme was added to the refolding buffer having hydrophobic protein and > 50% tag cleavage was observed. What could be the reason for not getting > 90% tag cleavage. I was wondering does the refolding buffer components especially 2M Arginine/2M Proline will interfere with enzymatic activity.
Yes, Arginine often inhibits the activity of proteases and hydrolases. I had used Arginine (50mM, 100 mM, 250 mM, 500 mM and 1.0 M) to suppress aggregation of proteins during thrombin-mediated cleavage of the GST fusion tag. Arginine (at > 0.1 M) interfered the activity of thrombin. As the concentration of arginine increased, the activity of the enzyme decreased.
The concentration of arginine in your case is very high. Moreover, the presence of other additives, such as Proline, may have additive or synergistic effect to inhibit the activity of the enzyme. You need to look for other additives that will prevent protein aggregation during refolding without interfering the activity of the enzymes.
I personally suggest you to use a high-throughput screening platform for identification of your buffer additives.
Dear Alemu Tekewe,
Thank you so much for the information.
Best regards,
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