Dear fellow researchgate scientists,
I hope this post finds you well.
In biochemistry laboratory classes we got about purifying proteins for which the structure and function are known, so it's easier to design a purification strategy.
Now, consider we want to purify a protein which has not yet been characterized. Should we consider the tissue from which it was extracted, and try to design an assay to understand its role? What if the protein in not an enzyme, how would you monitor its presence?
I would be glad if someone could share their input.
My kind regards
Dear André Antunes
To desing purification sttrategy the knowledge of the sequence and the possibility to produce the protein as recombinant protein is more essential than the function.
The sequence allow to predict if the protein topology (eg soluble, membrane) and desing a sintetich construct and expression and purification strategy (eg adding a cleavable tag for affinity purification).
To date thanks the avaialbility of powerfull bacterial, yeaat and mammalian expression sistem, the purification of a soluble protein is generally simple while the purification of a membrane protein is still challenging aince you need to found the right detergents able to mantanin the protien folded and not aggregate.
good luck
ciao
Manuele
Function could be usefull to check if the produced protein is active and folded.
Dear Dr. Manuele Martinelli thank you for the kind and clear reply,
So, for example, lets say I have a portion of eye cilliary cells. I homogenate the tissue, and procede towards differential centrifugation. In the supernatant sampled at the highest speed, I have several proteins, some of which I know, and others which I do not know.
Lets suppose I fractionate several proteins via chromatography, and I assay all the obtained samples. One of those corresponds to unknown protein. I would then analyse the sample with mass spectrometry, to determine the primary structure?
Then, I would seek to understand which genes code por that protein, and develop knockout models, to understand which function the protein might fulfill?
This is all very interesting, but very complicated!
My kind regards,
André Antunes
If you do not know the function, you cannot establish an assay unless you develop antibodies to the proteins. For example, Sds gels are great for separation of proteins ( but totally destructive of function). The individual proteins can be eluted and used for Ab production. This method becomes less reliable the more bands you have.
I have approached this problem myself by raising scores of monoclonal antibodies against a crude mixture and using them for immunoaffinity purification. This gives you pure proteins directly without the need for an assay, but still leaves the question of function.
You also have some useful tools then to study distribution and presence of antigens in other tissues, which may gives some clues. With pure proteins you can at least test for function. If you suspect you have enzymes you can add a range of substrates, but you need a lot of luck. I did get lucky!
Or you can take the molecular approach set out in the previous answer. Again it may provide clues but not necessarily a definitive function. However a molecular approach is more likely to be rewarded with useful data than the testing scores of substrates, only to find you never had an enzyme in the first place.
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