Improved tagging strategy for protein identification in mammalian cells.

ABSTRACT: The tagging strategy enables full-length endogenous proteins in mammalian cells to be expressed as green fluorescent fusion proteins from their authentic promoters.We describe improved genetic tools to facilitate protein tagging in mammalian cells based on a mobile genetic element that harbors an artificial exon encoding a protein tag. Insertion of the artificial exon within introns of cellular genes results in expression of hybrid proteins consisting of the tag sequence fused in-frame to sequences of a cellular protein. We have used lentiviral vectors to stably introduce enhanced green fluorescent protein (EGFP) tags into expressed genes in target cells. The data obtained indicate that this strategy leads to bona fide tripartite fusion proteins and that the EGFP tag did not affect the subcellular localization of such proteins.The tools presented here have the potential for protein discovery, and subsequent investigation of their subcellular distribution and role(s) under defined physiological conditions, as well as for protein purification and protein-protein interaction studies.

SUBMITTER: Bialkowska A 

PROVIDER: S-EPMC1250225 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature