Isolation of Endogenous Protein Complexes via Epitope Tagging in ES Cells
While most protein complexes are isolated through the over-expression of an exogenous tagged protein within cells, ideally complexes would be isolated with endogenous proteins as the target. To meet this need, Laboratory of Proteomics and Analytical Technolgies (LPAT) scientists are developing homologous recombination methods to incorporate epitope tagged proteins of interest directly into an organism’s genome. This ability would allow the complex being extracted to more accurately reflect the in vivo complex that exists within the cells.
LPAT scientists are taking two different approaches. The first approach is based on homologues recombination into specific side in the genome. A vector harboring the epitope and drug resistant gene will be flanked by 5’ and 3’ sequences homologues targeting side. Following selection the drug resistant gene is removed through flanking loxP sides and introduction of Cre. Final verification will be done through PCR amplification using primers specific to the epitope and the gene being targeted. The second approach relies on recombination rather than homologues recombination. It uses two vectors a targeting vector and a vector expressing the recombinase (FLPe), that are transfected into mES cells. The system that has been used with good success to modulate micro RNA expression. However, it has not been used to integrate specific epitope in frame with a gene of interest, therefore we might have to specifically adapt it for that purpose.