Hi
I am trying to express a fusion protein where one of the protein is a receptor protein and the other is a soluble protein. I have cloned them together but for some reason, I have not been able to express the fusion protein. I have tried making lentivirus and transduced mammalian cell line and I have had no luck with that either. Could someone provide their insights into constructing fusion proteins, using linkers, using tags and how to design these things with respect to solubility and flexible structure that it does not affect the normal folding of the 2 proteins in the fusion construct?
Thanks
What type of fusion are you doing? People frequently use maltose-binding protein as N-terminal fusion partner (assuming the N-terminus of your receptor is extracellular) - of course you need to make sure your constructs contains the appropriate signal sequence for your expression system. As C-terminal (intracellular) fusion partner, sfGFP may be useful, as it gives you immediate feedback on expression level and sub cellular localisation of your protein. Often several different receptor truncations, linker lengths and fusion partners have to be tried to find the combination that's best for your protein.
What type of fusion are you doing? People frequently use maltose-binding protein as N-terminal fusion partner (assuming the N-terminus of your receptor is extracellular) - of course you need to make sure your constructs contains the appropriate signal sequence for your expression system. As C-terminal (intracellular) fusion partner, sfGFP may be useful, as it gives you immediate feedback on expression level and sub cellular localisation of your protein. Often several different receptor truncations, linker lengths and fusion partners have to be tried to find the combination that's best for your protein.
How are you detecting expression of this fusion protein (or lack thereof :-)? Also, is this a construct targeted for the secretion pathway, or just cytoplasmic?
Hi Alejandro
The construct is just an expression vector, however, the protein of interest has a leader sequence at the N-terminus, thereby allowing for its appropriate targeting at the membrane of the cells. I have been using western blots and immunofluorescence to as my read outs for expression.
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