vector for expression of tag on cell surface - (Sep/24/2006 )

Hi,

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.

-genehunter-1-

Will you express a memberane protein or others?

-noirvan-

QUOTE (noirvan @ Sep 26 2006, 02:53 AM)

Will you express a memberane protein or others?

Either a transmenbrane or a protein that is membrane anchored with a lipid is fine.

-genehunter-1-

genehunter,

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.

-scolix-

Yes. scolix.

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.

-genehunter-1-

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,
Ceri

-Ceri-

Thanks for the info. I will look into it.

-genehunter-1-

hi,

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

tk

-tadzik-