I want to clone a viral membrane protein in pGAPZαA (Yeast expression vector) which have N terminal signal sequence during infection it transport it into host(mammalian cell) golgi body and where whole protein cleaved in 2 parts by Furin enzyme. pGAPZαA have α Secretory signal which help in transport recombinant protein outside of the cell.
Please suggest me some doubt on that
1- If I express same protein in pichia pastoris will it cleaved in golgi body into 2 part or not?
2- If I clone full length protein with its signal sequence just after α Secretory signal of pGAPZαA then both signal sequence will work together or not?
3- α Secretory signal of pGAPZαA will be able to export the protein outside the cell OR it will incorporates in Membrane of pichia pastoris due to presence of its trans membrane domain?
4- Can anybody give me reference related to my Query?
1- Probably not. As far as I know, there's no furin-like protease in yeast
2- No, they should not. Bear in mind that what you have in pGAPZaA is a propeptide; i.e., it has the signal peptide of the alpha factor, plus parts of the alpha factor that are sequentially chopped off during ER/Golgi transport by the KEX2 and STE13 proteases. In your case, you'd be left with a protein in the Golgi compartment with an uncleaved signal peptide.
3- Depends on the how your protein folds and assembles into the membrane in its native host, whether it requires interacting partners to do so that are present or absent in Pichia, etc. If your alfa factor-paramyxoviral protein fusion does not fold correctly, it might even never leave the Golgi (there is quality control machinery down the secretory yeast pathway that acts on the alfa factor).
4- Regarding furin cleavage, do a google scholar/pubmed search for flaviviral prM-E fusions in Pichia (those are cleaved by furin in mammalian hosts).
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