I am planning to clone Ca sensing receptor(CaSR) in EGFP vectors and overexpress in breast cancer cell line. I will use pEGFP-N1 and pEGFP-C2 expression vector. For pEGFP -N1 vector, GFP protein will remain fused with the C-terminal region of expressed CaSR protein. And for pEGFP -C2 vector, GFP protein will remain fused with the N-terminal region. My concern is that in such cases, functionality and cellular localization of CaSR may be altered or may be even not seen because of the fused GFP protein. I goal is to study interaction and co-localization of a partner protein of CaSR. I would greatly appreciate all kind of suggestions. Thank you.
The most important factor is that your cloning keeps in frame the sequence of the gene with that of the GFP. Also, it helps to have a short polylinker of a few glycines in between the two proteins, to help their separate folding.
You'll need to demonstrate that your fusions localize the same the endogenous protein. If you aren't certain about whether either terminus is involved in localization, you may as well try both fusions.
That said, there are many, many successful examples of this type of fusion, so you should just do it and not lose sleep over it. Do the localization control. If you have an activity assay, do that as well.
Thank you Prof. Eric J. Arnoys for you suggestion. I will use both the vectors and check for any changes in localization of native protein and N-terminal and C-terminal GFP fused protein.
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