I am looking for protocols for extraction of NATIVE unknown proteins from a plant source. The proteins from the specific plant are not reported in literature. At the moment I am using a phosphate buffer 0.1M, 3% PVPP + 1% bisulphite? My procedure right now involves blending, extraction, centrifugation, dialysis, freeze-drying. I am looking for ways to improve yield and quality of the proteins. The protein powder will be tested for techno-functionalities.
Dear Bilyana:
If you don't have any other previous information, the best way to start is using buffers that mimic somehow the cytoplasmic composition. Are you isolating total protein content? membrane proteins?
The procedure you mentioned sounds ok, but other parameters like salt concentration, presence of DTT, or different processes to break plant tissues like French press or sonication may improve the isolation.
https://www.ncbi.nlm.nih.gov/pubmed/23240604
Article The proteins of the grape (Vitis vinifera L.) seed endosperm...
Good luck,
Rosa María
Avoid freeze drying. Many proteins do not survive that. Instead, flash-freeze the dialyzed extract in aliquots and store them at -80oC,
Dear Rosa and Adam, thank you for your replies.
@Rosa, I do want to extract the total protein, yes. I have not found any literature on the cytoplasmic composition. What do you mean by buffers that mimic it? I have not added DTT since I want to keep the proteins in their native state and DTT denatures proteins by reducing their disulfide bonds. I will look further into pressing and sonification. Do you think that increasing the buffer molarity might increase the yield? Thanks!
@Adam, I need to produce a powder in the end, this is why I freeze-dry it. If can share better methods, I would like to hear them.
Dear Bilyana,
The simple protocol you've adopted seems good. I would suggest you vary the concentration of your buffer and/or change the type of buffer to see if that increases the amount of extracted protein. But then, did you do a protein profile of the plant sample using LC/MS?
If you must dry the protein, it would be worthwhile to explore different excipients and buffer conditions (salts, pH, reducing agents) to see what is best for preserving the protein's structure and activity. Some proteins can be dialyzed against distilled water and freeze dried without loss of activity, but this is often not the case. Sugars such as trehalose can be helpful in preserving the activity of freeze-dried proteins. Do not include anything that is a nonvolatile liquid, such as glycerol or some detergents.
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