Hello,
I am new to using Drosophila S2 cells for protein production. I would like to over-express a membrane protein gene (12 TMH predicted with intra-cellular N and C-terminii) in S2 cells.The gene has no signal sequence.
Q1. Should I add a signal peptide sequence at the N-terminus for membrane targeting in S2 cells? Any suggestions about the signal peptide sequence and the peptidase cleavage site? I found this signal sequence in a paper: MGAVLRSLLACSFCVLLRA (ref: Le et al., Molecular Biotechnology (2018) 60:31–40)
Q2. Also, which vector should I use? Would one of the pMT/V5/His-A,B,C vectors be a good place to start?
Q3. It has been suggested that for proteins with extra-cellular N-terminii, a BiP secretory sequence should be used to improve expression. Is there any suggested sequence/tag for proteins with an even number of trans-membrane helices with both N and C terminii on the intra-cellular side?
Thank you.
Hello Saif.
Your protein has already membrane-spanning domains. Upon overexpression, the proteins are synthesized in the ER, becoming transmembrane proteins in the ER membranes. Through normal cellular machinery, the proteins will be transported into cis-, mid- and trans-Golgi and then the final destiny to the cell plasma membranes by normal cellular process (secretory vesicles or whatever) because the protein does not have any delivery code to any organelles. You could use any designed commercial vectors with tag and recommended column.
Best wishes.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes