Lentiviral fluorescent protein expression vectors for biotinylation proteomics

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• In vivo biotinylation tagging facilitates protein complex isolation in mammalian cells.
• Escherichia coli BirA biotin ligase is key for in vivo peptide biotinylation.
• Previous methods lacked precise control over gene expression and biotinylation levels.

Purpose of the Study:

• To describe a modified in vivo biotinylation tagging methodology.
• To enable stable cell line selection using fluorescence-activated cell sorting (FACS).
• To isolate and characterize proteins associated with the leukemogenic transcription factor TLX1.

Main Methods:

• Development of a FACS-based binary in vivo biotinylation tagging system.
• Utilized lentiviral vectors for coexpressing tagged genes with yellow fluorescent protein (YFP).
• Employed a second lentiviral vector encoding a fusion protein of bacterial BirA and green fluorescent protein (GFP).

Main Results:

• Successfully established a system for stable cell line selection via FACS.
• Demonstrated precise control over BirA-mediated biotinylation and gene of interest expression.
• Applied the method to isolate and characterize proteins associated with TLX1.

Conclusions:

• The modified FACS-based binary system offers enhanced control for in vivo biotinylation tagging.
• This technique is valuable for studying protein complexes, especially when high expression is detrimental.
• The method provides a robust approach for isolating and characterizing protein interactions in mammalian cells.