I'm trying to express a human cytokine (belongs to the TGFb superfamily) in HEK 293-E cells for the purpose of purifying the protein in an active form (i.e. a disulfide-bonded homodimer) 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!
Edited by guyava, 10 July 2013 - 03:06 AM.