Improved tagging strategy for protein identification in mammalian cells.
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.


CONCLUSIONS
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.
Bialkowska
Agnieszka
A
Gene Therapy Program, Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA. abialk@lsuhsc.edu
Zhang
Xian-Yang
XY
Reiser
Jakob
J
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005091:Exons; D017930:Genes, Reporter; D005819:Genetic Markers; D005821:Genetic Techniques; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D008957:Models, Genetic; D012516:Osteosarcoma / Q000378:metabolism; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D005819:Genetic Markers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012313:RNA