vector for expression of tag on cell surface - (Sep/24/2006 )
Can anyone give me a suggestion for an expression vector that will allow me to present a tag motif on the cell surface to allow me to identify and possibly purify transfected cells? Thanks.
Will you express a memberane protein or others?
Either a transmenbrane or a protein that is membrane anchored with a lipid is fine.
I am sure u thought abt it, transfect GFP and sort using FACS or make stable cell lines.
Or may b I completely misunderstood u.
Our FACS costs me $100.00/hr and I have to wait in line all the time. I finally decided that I have to use magnetic absorption as an alternative.
It looks like some GPI anchoring domains are not very big. I am thinking about synthesizing it from scratch, if I can not get any available constructs . Thanks.
I think Miltenyi Biotech have a system using a truncated mouse MHC class I molecule (I'm sure they used to use mouse CD4) called MACSelect kit. Their vector for the MHC class I also has a multiple cloning site so you don't have to co-transfect. You can then purify the transfected cells with MACS microbeads for the MHC.
Realistically you could do this with any cell surface marker by co-transfecting the expression vector with the vector you want as long as you have a good monoclonal which works in flow cytometry for the marker. I did this in my old lab with a co-transfected rat CD2, an anti-rCD2 monclonal and anti-mouse IgG MACs beads. We got 80-90% positive purity following sorting from a 40% transfection efficiency. I guess the commercial kit should be easier/more efficient.
All the best,
Thanks for the info. I will look into it.
and what about using a His-Tagged membrane protein? will such tagging affect developing a protein to the membrane?
for example: consider GPCR tagged with poli-His in stable transfection...
will it work and if the protein will have a membrane localization? (does His-tag influences mebrane localization signal?)
thanks ina advance for your answers