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Recombinant expression of cytokines in mammalian cells without the propeptide re

propeptide cytokines mammalian cells 293 HEK

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#1 guyava

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Posted 23 July 2013 - 05:46 AM

I posted this in another forum with no responses. Posting again here:

Hi,

I'm trying to express a TGFb superfamily human cytokine in HEK 293-E cells for the purpose of purifying the protein in an active form from the medium (so the protein should be secreted). Like most cytokines, this protein has a signal peptide that renders it secreted, a propeptide and a mature domain. When I monitor the expression, I always get a lot of the propeptide-mature form (the propeptide is not cleaved and is expressed in the final protein) whereas the mature form is expressed in minute amounts. What would be the effect of constructing a gene that would lack the endogenous signal-peptide and propeptide - for example, an Igk signal peptide fused to the mature domain of the cytokine (i.e. no propeptide, and replacement of the original signal peptide with a supposedly stronger one)? I'm worried that this would somehow ruin the activity or the dimerization. Is there any "rule" that applies to all cytokines?

Thank you in advance!

#2 dimensionsbio

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Posted 28 September 2013 - 01:16 PM

Hi guyava.  In my experience, using a non-native secretion signal should not be an issue as long as the cell line recognizes it.  Not including the propeptide sequence should not affect dimerization either since dimerization will occur between the two mature 12.5 kDa molecules in TGFb – nothing to do with the propeptide.  The activity will have to be determined experimentally.







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