I knocked down wnt5a gene by using siRNA against Wnt5a and observed that wnt5a and its receptor ROR2 expression decreased but adding exogenous recombinant wnt5a protein to the cells treated with siRNA wnt5a.can not revert RORs expression what can be the resposible response to this observation?
because of misfolding - a constant feature of recombinant proteins
Dear Ghobadi,
As already suggested that the protein may not be properly folded to be functional. It would depend on the host in which you have overexpressed and purified the protein. Mammalian proteins expressed in some other hosts like bacteria will not have the same disufide linkages, no similar chaperones for folding and hence the protein won't be active. If its a eukaryotic protein, then it must be expressed in some eukaryotic host. The codon bias also varies in different organisms, but at least the folding can be expected to be similar. Express the protein in a compatible system, confirm its secondary structure (in CD spectra may be), check for the activityand and then use it for your experiments.
Hope it helps!
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