Hi Mahamoud,

PBS has a salt concentration around 150mM and pH ~7.5.  I've used Sarkosyl extensively as a solubilising agent and find it very useful, however as you've said sometimes the proteins are not stable once the Sk is removed (if removal is necessary for the end use).

First question regarding the stability : what is the pI of your protein?  If the pI is naturally close to the numerical pH of standard PBS, it could be that you need to modify your dialysis buffer to something that the protein may be more compatible with - a lot of proteins I work with are very compatible with pH8.0 or 8.5.

For instance : instead of pH7.5 - would pH8.5 or 6.5 be more suitable?  Could you use a buffering agent other than Phosphate - such as 50mM HEPES?

Secondly, try increasing the average salt concentration to 300mM, or 500mM?  Sometimes this is sufficient to retain solubility, providing again it is compatible with your end use.

Thirdly - is it possible to include other components in the buffer than may help with protein stability?  i.e. low % glycerol etc?

Fourthly - Arginine and related compounds are often included in buffers to retain solubility - again, if it is compatible with your end use, 0.1-0.5M Arginine can help.

Please come back to discuss these ideas further if they are of any interest to you.

Good luck!

Detection and prevention of protein aggregation before, during,

and after purification

Sarah E. Bondos* and Alicia Bicknell1

Abstract

The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS–PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include

monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins. 

Article Detection and prevention of protein aggregation before, duri...

Dear Mahamoud,

I am not aware of the circumstances in which your protein was extracted. But, to the best of my knowledge, when urea is used during the purification of inclusion bodies protein, the purified protein should not be frozen (only stored at +4 degre), if not it will be aggragated. To avoid this, there is need to proceed to a complete dialysis (for urea-free/lower) and refolding of your protein.

Good luck !

Protein Aggregation

A key challenge in recombinant protein production is to maintain and store the target protein in a soluble and stable form. Protein aggregation can compromise protein function and thus it is necessary to overcome this challenge to generate functionally active protein.

Aggregates can be categorised as either “insoluble” (able to be removed by centrifugation or filtration) or “soluble” (not easily separated from native protein). Techniques such as analytical size exclusion chromatography (SEC), dynamic light scattering (DLS) and ultracentrifugation play an important role in identifying soluble aggregates.

Aggregation can occur at any stage of the production pipeline:

• Protein Expression (e.g. inclusion body formation)
• Protein PurificationCell lysis and extraction
• Chromatography
• Buffer Exchange
• Concentration
• Storage

A number of strategies can be employed to overcome aggregation and promote protein stability.

The use of fusion tags, such as maltose binding protein (MBP) or thioredoxin (Trx), can impart solubility on proteins expressed heterologously in E. coli. Modifying expression culture conditions (e.g. reducing temperature) may also improve solubility by promoting correct folding.

Buffer conditions can be optimised to improve the target protein’s solubility during purification. Additives such as reducing agents (e.g. ß-mercaptoethanol, DTT), chaotropes (e.g. urea, guanidium-HCl), kosmotropes (e.g. ammonium sulphate, glycerol), detergents (e.g. tween, CHAPS), amino acids (arginine, glutamine) and ligands or cofactors (protein-dependent) can be used in low concentrations to stabilise the target protein’s native structure. Additionally, buffer pH and ionic strength also influence protein stability. Therefore it is often necessary to screen an array of buffer conditions and additives to determine the optimal buffering environment for the target protein. Once these stabilising conditions are known, they can be implemented throughout the purification process.

High protein concentration can compromise protein stability. Consequently, it may be necessary to maintain a low protein concentration by increasing the sample volume during lysis and chromatography. In situations where a high final protein concentration is required, stabilising buffer components can be included to avoid protein aggregation and maintain solubility.

Many proteins are unstable at 4˚C for more than a few days, so the preferred strategy is to store purified protein at -80˚C. However, subjecting proteins to repeated freeze-thaw cycles often leads to protein precipitation, so it is good practice to scout stability in advance. Buffer conditions that favour protein solubility at 4˚C may not necessarily prevent aggregation during freeze-thaw. Glycerol is often added to the protein sample as a cryoprotectant.

Other practices that reduce the propensity for proteins to aggregate include:

• Performing all purification steps at 4˚C
• Minimising sample handling
• Avoiding time delays between purification steps
• Reducing exposure to air-liquid interfaces (e.g. by avoiding bubble formation)

https://www.uq.edu.au/pef/content/protein-aggregation

I would like thank all of you for the valuable suggestions.

I tried changing the salt concentration ranging from 300 to 500nM as suggested by Ali and under several PH as suggested by Robin. Also I tried PEG 400, 6000 and 8000 with glycin or glycerol.

Finally< Conducted a cytotoxicity test of N-lauroylsarcosine on several cell lines (adherent cells) in order to know the minimal concentration of N-Lauroylsarcosine that can exhibit a toxicity. Then my protein is now soluble in that minimal concentration (between 0.01-0.02%).