I want to construct a plasmid in this sequence order (from N to C): a protein X--GnRHR (it is a GPCR)-- a reporter gene. I want to express this fusion protein on the cell membrane. But protein X is not typical cell-membrane-expressing protein. I also used SignaIP to predict the signaling peptide of GnRHR, but the result gave no signaling peptide. Then I discovered a material saying use from N terminal of GPCR to the 1st helix will help the fusion protein to express on the cell membrane. So I carry out the plan: N terminal~ 1st helix of GnRHR --protein X -- GnRHR -- reporter protein. (Honestly, I forget to trace back the material to persuade my supervisor.)
I hope to receive comments or if there are better solutions. Thanks a lot, all!
Junhui, when there is no signal sequence recognized by computational algorithms, but the protein is known to get into the right pathway, you're on the safe side.
You might want to try to identify the signal sequence either by mutation experiments or by tryptic digests and mass spec, if you are able extract your protein of interest with an antibody.
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