Hi there,
I am carrying out crystallisation experiments for lysozyme. The method is the following: dissolve 73.6 mg/ml lysozyme (lyophilised or crystalline powder) in 0.1 M sodium acetate buffer, and dissolve 80 mg/ml NaCl in 0.1 M sodium acetate buffer. Mix both solution with a ratio of 1:1 to achieve a final crystallisation composition of:
- 0.1 M sodium acetate buffer
- 40 mg/ml NaCl
- 36.8mg/ml lysozyme
-->the supersaturation ratio should be around 10
These crystallisation condition led previously to crystallisation: solution stayed clear after mixing all components and well-built crystals were visible with the first 3 hrs.
However, I am using new lysozyme powder batches (lyophilised or crystalline powder) and immediately after mixing the solutions (NaCl with lyophilised or crystallised lysozyme), the solution turns cloudy. Instead of crystals, after 1-3 hours only small precipitate or aggregated proteins are occurring. I just purchased the new lysozyme powders very recently but cannot explain why they don't crystallise out anymore?
Anybody an idea?
Many thanks, Frederik
Hi Frederik Link,
Hard to tell what is going on since lysozyme crystallizes very easily.
First, I would dissolve the lysozyme and leave it for a while (maybe sonicate a little?) to ensure complete dissolution.
Maybe measuring the pH of your mixture could indicate that something is off; if the pH is too far from ~5 (the pH of your sodium acetate buffer), this could possibly lead to the precipitation in the drops.
Another idea is to check the lysozyme concentration by absorbance. If, by accident, you are using a too higher concentration, this could lead to precipitation.
I would also run an SDS-PAGE gel (or MS, even better) to check the purity of your lysozyme powders.
Best,
Italo Cavini
Post-doc at the Biophysics and Structural Biology Laboratory
Physics Institute of São Carlos (IFSC), University of São Paulo - Brazil
Frederik Link
Various additives greatly influence the formation of nuclei and crystallization of substances. Therefore, look in the literature for obtaining and purification of lysozyme. One of these works
https://www.tandfonline.com/doi/full/10.3109/13880200903196859
There are a lot of such works.
~ 0.20 micron syringe filter all solutions including your lysozyme protein. The pH of the acetate buffer should be about 4.8. My instructor who is an experienced crystallographer is absolutely convinced that some commercial lysozyme is crystallization grade and some lysozyme powders are not crystallization grade. Just because it possesses enzymatic activity doesn't mean it is high enough quality to crystallize. He can crystallize some batches of lysozyme and not others.
Also the structure of lysozyme is already known to sub angstrom resolution so that is not much to learn about it now.
Dear Frederik,
I have observed something similar from the experiments of a a lab colleague (especially at such high supersaturation levels). Even within the same commercial brand, the lysozyme batches might slightly differ. My proposal is to always filter all the solutions (as mentioned in a comment above) and dialyze the protein. There are plenty of dialysis procedures, but let me know if you have questions.
Regards,
Joana
Dear Adron Ung ,
thank you for the comment on the pH. The pH of my buffer is 4.6. I also bought a brand new lyophilised lysozyme sample from sigma Aldrich and the same cloudiness occurred (SA buffer pH 4.6). Hence I am sure that the lysozyme should be alright
Dear Joana Ferreira ,
i think dialysis is similar to filtration? Filtering the protein solution through a centrifugal filter for example? To do so, and lysozyme being ~14 kDa in size, which filter, dialysis procedure would you recommend?
Best wishes
Frederik
Hi Frederik Link
It is possible that certain salts (or even additives) might be present in the lyophilized lysozyme, which might not be removed by filtration (but possibly by dialysis).
Normally, all my solutions are filtered using syringe filters (Puradisc FP 30 mm, cellulose acetate, 0.2 μm) (Sigma-Aldrich, Whatman) to remove large aggregates.
Lysozyme should be dialyzed against the buffer solution (in your case, sodium acetate) overnight and gently stirred at 4 ºC. A standard grade regenerated cellulose dialysis membrane (Spectrum Labs) with a molecular weight cut-off < 14 kDa was selected based on the molecular weight of lysozyme (~ 14 kDa).
Hope this helps. I followed this dialysis procedure: https://www.sciencedirect.com/science/article/abs/pii/0003269787902211.
Best wishes,
Joana
Dear Frederik
Lysozyme crystallization is very sensitive to temperature. If all materials were dissolved and mixed immediately after being taken out of the refrigerator, the temperature of the crystallization solution is not arrived to room temperature, a lot of small crystals or precipitate will occur.
Besides, we found that different production batch of lyophilised or crystalline powder have different crystallization behavior especially of triple recrystallized lysozyme. Maybe different batchs of triple recrystallized lysozyme have different kinds or amounts impurities(dimer, multimer, or additives) . Well, six recrystallized lysozme have good reproducibility. So, the same batch number lysozyme is recommended for crystallization experiment.
Good luck,
YongMing
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