In certain cases, it is not possible to obtain functionally active proteins using the E. coli expression system. In such an event, it would be necessary to carry out protein expression in mammalian cells. The purified proteins can be used for enzyme assays or high throughput screening assays to the discretion of individual investigators. Similarly, stable cell lines expressing GFP-tagged proteins can also be extremely useful in tracking the intracellular distribution of a protein.
To develop stable cell lines, the Protein Core will assist investigators in carrying out the following services:
A) Make a large-scale prep of the plasmid expressing the protein of interest.
B) Transfect mammalian cell with the plasmid.
C) Select transfected cells with antibiotics.
D) Obtain stable clones expressing the protein of interest.
E) Expand the clones and freeze down aliquots.
F) Purify the recombinant protein expressed by the stable cell lines.
The purified recombinant proteins can be used for drug screening or for high-throughput screening of compounds. The Protein Core will facilitate the interface with the Drug Discovery Center to provide the needed proteins for investigators.
Tracking GFP-Bax movement during apoptosis. MCF-7 cells stably expressing GFP-Bax and mitochondrially targeted red fluorescent protein (Ds-Red Mito) were treated with C6-pyridinium ceramide. The translocation of GFP-Bax to mitochondria was visualized by fluorescence microscopy.
Specialty Protein Services Provided