Tandem affinity purification of protein complexes in mouse embryonic stem cells using in vivo biotinylation

Abstract

In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed a protein-protein interaction network. This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for in vivo biotinylation system setup in mouse ES cells, and affinity purification of multi-protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS-PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification.

Figure 1. Establishment of a biotinylation system in J1 ESCs

A stable ESC line expressing the bacterial BirA enzyme was first established by transfection with a BirA-expressing plasmid bearing the neomycin resistance (neo^r) gene and G418 selection; A second plasmid containing cDNA encoding a transcription factor (TF) of interest with an N-terminal Flag-biotin dual tag (FLBIO) and a puromycin resistance (puro^r) gene was introduced and cells selected with puromycin. The resulting stable line is resistant to both G418 and puromycin and express FLAG-tagged, biotinylated TF (^FLBIOTF) that can be immunoprecipitated by anti-FLAG and streptavidin antibodies/beads.

Figure 2. A summary of the procedure for tandem affinity purification of multiprotein complexes in mouse ESCs

Following establishment of BirA-only and BirA+^FLBIOTF expressing ES cell lines, immunoprecipitation is performed using anti-FLAG M2 agarose (FLAG-IP). The bound material are eluted with FLAG peptide and further purified by streptavidin affinity capture. The purified protein complexes are fractionated on SDS-PAGE, and subjected to LC-MS/MS to identify components of the protein complexes.

Figure 3. Examples of Western blot analyses

(A) A Western blot with anti-V5-HRP to detect BirAV5 expression. (B) A Western blot using native antibody against the TF of interest to detect both endogenous and biotinylated TF proteins. The sub-endogenous level of the biotinylated transcription factor (^FLBIOTF) and the expression level of the endogenous protein (^endTF) are indicated. “NS” denotes non-specific signals.