I recently purchased an expression construct from GenScript in order to overexpress a FLAG tagged protein in mammalian cells. The construct is based on pcDNA3 and has a Met, the FLAG epitope, and then the protein of interest beginning with the second amino acid. My concern is that the protein of interest in a transmembrane protein. Might the N terminal tag interfere with transmembrane insertion? Could the tag be removed by signal peptidase? I am asking, of course, because I have not seen evidence of overexpression in 293T cells by western blot with M2 anti-FLAG. I lyse my cells directly in SDS sample buffer and have loaded samples with and without boiling as I know that can cause problems with TM proteins. I also transfected a different protein as a positive control and it overexpressed very well. Any advice would be appreciated!
It depends on the type of membrane spanning protein you are working with, but the answer is often "yes", the tag can cause problems, it can impair protein function, it can be cleaved proteolytically and it can even flip membrane orientation and cause malfolding/degradation of your protein.
1) If it is a type I single membrane spanning protein, it will have an N-terminal signal peptide for translocation of the N-terminus into the lumen of the ER and further downstream in the sequence a transmembrane domain (also known as stop transfer sequence) to fix the protein in the membrane (C-terminal tail is cytosolic). In this case, putting an epitope tag between the start (methionine) codon and the second codon would not work, because 1) the tag might influence the functionality of the signal peptide and 2) it will be cleaved together with the N-terminal signal peptide in case the functionality has not been compromised. In this case, you can insert the tag between the signal peptide and the beginning of the mature part of the protein or you tag the C-terminus, I would try both and see which works best.
2) If it is a type II membrane spanning protein (single span), you can try tagging either the N-terminus or the C-terminus, it may work or it may not, it always depends on the protein.
3) If it is a multiple membrane spanning protein, you need to make a prediction first to check where the transmembrane domains are and how much N-terminal and C-terminal tails you have. The membrane orientation of multiple membrane spanning proteins is often determined by the charges flanking the first transmembrane domain, with positive charges often found on the cytosolic side. I have had a case where an N-terminal fusion caused a 7 transmembrane domain protein to flip orientation, the N-terminus was suddenly in the cytosol when it should have been in the lumen, and the fusion protein was misfolded, trapped in the ER and rapidly degraded.
I can only give specific advice if you send me the primary amino acid sequence of your membrane protein.
:)
Hi there,
Effect of the tag will vary with the actual topology of the transmembrane protein: if the Nter of the membrane protein is cytoplasmic then the presence of the tag shouldn't affect membrane insertion whereas if the Nter is extracellular is means the tag should have to cross the membrane first (ie prior the signal peptide) which is less than probable (signal peptide should be upstream of the tag sequence in the gene in order to get the right process).
It depends on the type of membrane spanning protein you are working with, but the answer is often "yes", the tag can cause problems, it can impair protein function, it can be cleaved proteolytically and it can even flip membrane orientation and cause malfolding/degradation of your protein.
1) If it is a type I single membrane spanning protein, it will have an N-terminal signal peptide for translocation of the N-terminus into the lumen of the ER and further downstream in the sequence a transmembrane domain (also known as stop transfer sequence) to fix the protein in the membrane (C-terminal tail is cytosolic). In this case, putting an epitope tag between the start (methionine) codon and the second codon would not work, because 1) the tag might influence the functionality of the signal peptide and 2) it will be cleaved together with the N-terminal signal peptide in case the functionality has not been compromised. In this case, you can insert the tag between the signal peptide and the beginning of the mature part of the protein or you tag the C-terminus, I would try both and see which works best.
2) If it is a type II membrane spanning protein (single span), you can try tagging either the N-terminus or the C-terminus, it may work or it may not, it always depends on the protein.
3) If it is a multiple membrane spanning protein, you need to make a prediction first to check where the transmembrane domains are and how much N-terminal and C-terminal tails you have. The membrane orientation of multiple membrane spanning proteins is often determined by the charges flanking the first transmembrane domain, with positive charges often found on the cytosolic side. I have had a case where an N-terminal fusion caused a 7 transmembrane domain protein to flip orientation, the N-terminus was suddenly in the cytosol when it should have been in the lumen, and the fusion protein was misfolded, trapped in the ER and rapidly degraded.
I can only give specific advice if you send me the primary amino acid sequence of your membrane protein.
:)
Thanks so much for taking the time to provide thoughtful answers! Clearly even if I do see overexpression, there is no guarantee that the protein will be localized and folded correctly.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques