Methodology article

Improved tagging strategy for protein identification in mammalian cells

Agnieszka Bialkowska, Xian-Yang Zhang and Jakob Reiser^*

• * Corresponding author: Jakob Reiser jreise@lsuhsc.edu

Author Affiliations

Gene Therapy Program, Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA

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BMC Genomics 2005, 6:113 doi:10.1186/1471-2164-6-113

Published: 4 September 2005

Abstract

Background

The tagging strategy enables full-length endogenous proteins in mammalian cells to be expressed as green fluorescent fusion proteins from their authentic promoters.

Results

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.

Conclusion

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.