Hi all,
I am struggling with expressing a viral glycoprotein in EXPI293 cells, I have done some mutations on my protein to trap it in a perfusion form, the postfusion form expresses naturally so, I had to do some calculated mutations which has worked for others in recent publications. But, I am unable to express it. My protein has a C-terminal doublestep tag and the wild type expresses really well but, there is no expression of the mutants. Can you help me trouble shoot, I trasnfect at 3million cells and harvest after 4 or 5 days and follow up with a streptactin XT based purification and confirm with antistrep western blot of the elutes with my wildtype as controls(which express). How to trouble shoot and does an other tag help with expression, where is my protein in the cell?
Your mutations can make your glycoprotein unstable or misfolded depending on your expression conditions which are not exactly the same as in the publication. Trying to improve the good refolding of your protein, it is difficult to advise you without knowing more.
I have tried several mutations, startign with some incorporation of disulphide bridges to trap the domains and even those did not work, my query, how to proceed in such situations where there is no expression. Of course, I intend to change my mutation strategy if that is the problem.
Dear Subhash Narasimhan
are you perfroming extracellular expression by addition of leader peptide or intra cellular expression?
best
Manuele
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA