Identifying "bottlenecks" in heterologous expression

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pkd
pkd's picture
Identifying "bottlenecks" in heterologous expression

Hi guys,

I'm a student looking for some advice on this theoretical problem.

Problem:

I want to express a mammalian membraneprotein (= a heterodimer) in the yeast S. cerevisiae.

I have already constructed an expressionvector and transformede my cells.

Now I want to identify possible "bottlenecks" in the expression - which methods would you use to answer this???
I'm just looking for a few simpel ones

Fraser Moss
Fraser Moss's picture
If you add a fluorescent tag

If you add a fluorescent tag or an epitope that can be recognized by a commercial antibody you could study the trafficking of the protein and co stain with organelle markers to look at where in the cell the protein aggregates or traffics under normal and various other conditions.

For instance if your protein is a heterodimer as you say, if you omit one of the subunits you will be able to visualize how it how that effects the expression and trafficking of the other. If you introduce mutations into one or the other of the subunits you could perform similar experiments to see their effects.

Fraser Moss
Fraser Moss's picture
you can also perform pulse

you can also perform pulse chase experiments to look at the half-life of the expressed channel in your cells