Improving protein solubility & long-term stability

[nmrlearner]

Has anybody successfully used the following method?

A simple method for improving protein solubility and long-term stability.

Golovanov AP, Hautbergue GM, Wilson SA, Lian LY.

Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, P. O. Box 88, Manchester M60 1QD, UK. a.golovanov@umist.ac.jp

J Am Chem Soc. 2004 Jul 28;126(29):8933-9.

Abstract:

Increasing a protein concentration in solution to the required level, without causing aggregation and precipitation is often a challenging but important task, especially in the field of structural biology; as little as 20% of nonmembrane proteins have been found to be suitable candidates for structural studies predominantly due to poor protein solubility. We demonstrate here that simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer can dramatically increase the maximum achievable concentration of soluble protein (up to 8.7 times). These amino acids are effective in preventing protein aggregation and precipitation, and they dramatically increase the long-term stability of the sample; additionally, they protect protein samples from proteolytic degradation. Specific protein-protein and protein-RNA interactions are not adversely affected by the presence of these amino acids. These additives are particularly suitable for situations where high protein concentration and long-term stability are required, including solution-state studies of isotopically labeled proteins by NMR.

[alan.robot]

Yes, I read the same paper and tried it with favorable results. I am working on a ~10kDa RNA binding protein from Drosophila which would precipitate after only 3 days in the NMR. After optimizing the buffer conditions it would last ~ a week but still slowly precipitate. I tried 50mM Arg/Glu and now my sample is happy for several weeks! Still have really bad s/n because of all the salt, though (if I go lower than 100mM KCl it aggregates, and the Arg-HCl and K+ Glu salts are really driving up the effective salt concentration). All that and parts of protein seem to be exchange-broadened But at least it lasts long enough to do most triple resonance experiments now.


Cheers,
Alan

P.S. Like the paper says it's important to add the arg/glu when the sample is fairly dilute, that is before the final concentration. Also, prepare some D2O with 50mM arg/glu to avoid cloudiness when adding the 10% to your sample.

[nmrlearner]

Originally posted by alan.robot@Sep 14 2005, 09:19 PM
Yes, I read the same paper and tried it with favorable results.* I am working on a ~10kDa RNA binding protein from Drosophila which would precipitate after only 3 days in the NMR.** After optimizing the buffer conditions it would last ~ a week but still slowly precipitate.* I tried 50mM Arg/Glu and now my sample is happy for several weeks! Still have really bad s/n because of all the salt, though (if I go lower than 100mM KCl it aggregates, and the Arg-HCl and K+ Glu salts are really driving up the effective salt concentration). All that and parts of protein seem to be exchange-broadened* ** But at least it lasts long enough to do most triple resonance experiments now.
Cheers,
Alan

P.S.* Like the paper says it's important to add the arg/glu when the sample is fairly dilute, that is before the final concentration.* Also, prepare some D2O with 50mM arg/glu to avoid cloudiness when adding the 10% to your sample.

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Thanks for your feedback, Alan! It is nice to know that this method works not only for the authors.

Still have really bad s/n because of all the salt, though (if I go lower than 100mM KCl it aggregates, and the Arg-HCl and K+ Glu salts are really driving up the effective salt concentration).

Does the protein precipitate with or without Arg-HCl and K+ Glu when you go below 100 mM KCl? If without, may be 50 mM Arg-HCl and K+ Glu can replace 50 mM KCl?

All that and parts of protein seem to be exchange-broadened* **

Well.. sounds like an RNA binding protein. You may want to try to change the exchange behavior by going to a different field-strength or/and to use CPMG in HNCA and other backbone assignment experiments.

Mark

[pedro_23]

Could someone give us more feedback about this method. Heard that it doesnt work that well.
Im trying to to do some NMR studies in solution, and im looking for some buffers that allow my sample to be stable along one week.

cheers,

[pedro_23]

Could someone give us more feedback about this method. Heard that it doesnt work that well.
Im trying to to do some NMR studies in solution, and im looking for some buffers that allow my sample to be stable along one week.

cheers,