truncated protein under normal conditions. - it is possible? (Jan/15/2007 )

we have found on 2DE of neurons that truncated form of our protein of interest is upregulated upon tropic factor deprivation. however there is a basal level of this truncated form on 2DE of normal neurons . My professor says it comes from the cell lysis and other preparations that we used. does it mean that those are only artifacts and truncated protein can't be found under normal conditions (untreated neurons)? thank you for your input on this.

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Hi Kathy,
What are your lysis conditions again? Have you tried different methods of lysing the cells, and if so, do they all give the same response? (That will be a clue as to whether the problem is in your protocol, or if it's a real phenomenon)

What's the difference between the full-length and the "truncated" forms of the protein? Could it be some kind of signal peptide/inactivating peptide?

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swanny, thank you for your reply, what is signal peptide/inactivating peptide? the difference between native form and cleaved one is around 2 KDa . and we tried only one lysis condition. so i dont know whether it is from the protocol or not but i am interested in case if not, is it possible to happen actually?

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Hi Kathy.
I had quick look on PubMed, and found one paper that describes what I'm talking about, I think. J Bioenerg Biomembr. 2002 Aug;34(4):307-15. is the reference.

If the difference between the two forms is 2 kDa, that could well be a small domain, about 20 residues long. There are a few examples of C-terminal domains acting as inactivation domains for transmembrane ion channels, such as this J Cardiovasc Electrophysiol. 2006 May;17 Suppl 1:S21-S25, and I suppose that was what I was thinking of when I asked the question. I have no idea if the same idea holds for intracellular proteins.

As for lysis methods, I tried a few during my PhD, and I found the best results came from freeze-thawing in the presence of protease inhibitors. It's very gentle (does anyone know of a gentler method?) and reasonably quick.

All the best.
swanny

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Perhaps rather than being a cleavage product of your full-mass protein, the "truncated" protein is a product of alternative splicing. Removal of an exopn could cause such a mass shift in the protein product. Why doe you think it is a truncation (like a cleavage product) and not an internal deletion (lake an alternative spliced form of the protein)? It is very reasonable that you might have a truncated protein; I am just pointing out a possible alternative hypothesis.

-Jon

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thank you very much for your answers.

so you are saying that two forms of my protein exist (maybe) and each one is produced from different mRNA and has a different function. is that what you are saying?

well, i dont know.... on 2D there is some intensity of the spot that i think is truncated even without any treatment. after treatment this spot's intensity is increased.

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highly interesting question, supervisors try to push a project straight forward (in the sense what they have come up in their grants) maybe your "problem" was not intended; I recommend to check what you have found

Is the truncated form also to detect in 1D electrophoresis (risk of proteolysis is lower as for 2D)?

Try to get the gene of full length protein, transfect/infect and check if a also the truncated form is coexpressed (if yes, hint to splice variant)

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