Lentiviral fluorescent protein expression vectors for biotinylation proteomics.

In vivo biotinylation tagging, based on a method in which a protein of interest is tagged with a peptide that is biotinylated in vivo by coexpression of Escherichia coli BirA biotin ligase, has been successfully used for the isolation of protein-protein and protein-DNA complexes in mammalian cells. We describe a modification of this methodology in which cells stably expressing the tagged gene of interest and the BirA gene can be selected by fluorescence-activated cell sorting (FACS). We recently implemented this approach to isolate and characterize proteins associated with TLX1, a homeodomain transcription factor with leukemogenic function. The modified technique utilizes two components: a lentiviral vector coexpressing the gene of interest containing a biotinylation tag on a bicistronic transcript together with a downstream yellow fluorescent protein (YFP) gene; and a second lentiviral vector encoding a fusion protein composed of bacterial BirA linked to the green fluorescent protein (GFP). This FACS-based binary in vivo biotinylation tagging system allows precise control over the levels of BirA-mediated biotinylation as well as the expression of the gene of interest, which is especially important if high-level expression negatively impacts cell growth or viability.