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.
Department of Anatomy and Regenerative Biology, The George Washington University Medical Center, Washington, DC, USA.
Irene
I
Riz
Teresa S
TS
Hawley
Robert G
RG
Hawley
D000818:Animals; D019921:Biotinylation / Q000379:methods; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D045744:Cell Line, Tumor; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation; D023061:Gene Order; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D040901:Proteomics / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D018398:Homeodomain Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C071250:TLX1 protein, human; D049452:Green Fluorescent Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli