Introduction
Traditionally, protein interactions have been identified using a variety of methods including yeast two hybrid screens (Y2H), coimmunoprecipitation (CoIP) using antibodies against endogenous proteins or epitope tags, and capturing complexes using affinity tags(1)
. Although Y2H is a powerful high-throughput in vivo screening tool, it can only identify binary interactions and is plagued by high rates of false positives. Antibodies are attractive for the isolation of endogenous multi-protein complexes, but they can lack specificity, sensitivity and availability. Both epitope tags and affinity tags can be used as an alternative to antibodies when antibodies do not perform well, or the protein of interest associates weakly or existes at low levels in the cell. For all these approaches, complex capture relies upon efficient binding to an affinity resin. While the HaloTag® system is similar in that it is based on a protein fusion tag, its rapid, covalent and irreversible binding sets it apart from other affinity tags. These properties increase the chances of capturing protein complexes and retaining them after capture. In addition, the lack of an endogenous equivalent of the HaloTag® protein in mammalian cells minimizes the chances of detecting false positives or nonspecific interactions.
The HaloTag® Mammalian Pull-Down and Labeling System
The HaloTag® Mammalian Pull-Down and Labeling System uses the HaloTag® protein tag, which can be genetically fused to any protein and mediates irreversible binding to the HaloLink™ Resin(2)
. A HaloTag® fusion protein can be expressed in mammalian cells either transiently or stably and, after it incorporates into its respective complexes, can be used as bait to capture in vivo interacting protein partners. After cellular lysis the HaloTag® fusion protein, bound to its interacting protein partners, is captured on the HaloLink™ Resin and gently washed. For analysis, the interacting protein partners can be eluted using denaturing elution buffers such as SDS or urea, or the protein complex can be removed from the resin using TEV (Tobacco Etch Virus) protease (Figure 1).
When eluting the fusion protein with SDS or urea, the HaloTag® fusion protein remains covalently bound to the resin. This results in release and enrichment of interacting protein partners only, and could be particularly advantageous when performing mass spectrometry analysis, where abundance of the bait protein has the potential to interfere with the identification of less abundant proteins in the sample. With TEV cleavage, the entire protein complex containing the untagged bait protein is recovered, which could be useful when performing functional studies. With either elution method, the recovered complexes are suitable for analysis by a variety of methods including SDS PAGE, Western blotting, mass spectrometry, and, in the case of TEV cleavage, functional studies.
The HaloTag® TMRDirect™ fluorescent ligand provided with the system is useful for measuring HaloTag® fusion protein expression and binding efficiency without an antibody. It is also ideal for performing complementary studies looking at protein cellular localization in live or fixed cells, protein trafficking and protein turnover(3)
.
Characterization of protein complexes and cellular localization of p65
To demonstrate the ability to capture binary and tertiary complexes using the system, we cloned one of the members of the NFκB family of proteins, p65 (RelA), into the HaloTag® Flexi® vector pFC14, and expressed the protein as a C-terminal p65-HaloTag® fusion protein either transiently in HeLa cells or stably in HEK 293 cells. Control samples expressing the HaloTag® protein alone were used for the pull-down experiments. NFκB consists of five structurally related subunits that can form various homodimeric and heterodimeric complexes: NFκB1 (p50; p105); NFκB2 (p52; p100); RelA (p65), RelB and c-Rel(4)
. These complexes are further regulated by interaction with IκB proteins (inhibitors of κB; (5)
(6)
). Our results show that all the expected interacting partners were recovered and identified only in the experimental samples containing the p65-HaloTag® fusion protein (Figure 2). Endogenous p65 also was detected, indicating the p65-HaloTag® fusion dimerized with the endogenous counterpart. A reciprocal pull-down experiment using an IκBα-HaloTag® construct yielded similar results (data not shown). Additionally, U2OS cells stably expressing the p65-HaloTag® fusion protein were labeled using the HaloTag® TMRDirect™ fluorescent ligand to confirm proper cytoplasmic cellular localization of the p65-HaloTag® fusion protein (Figure 3).
Capture of the higher order structural complex Nup107–160
As an example of a more complex system, we chose the nuclear pore complex (NPC), the supra-molecular structure embedded in the nuclear envelope that is responsible for the bidirectional transport of macromolecules between the cytoplasm and the nucleus(7)
. We performed pull-down experiments with two of the members of the Nup107–160 sub-complex. We used HeLa cells expressing N-terminal HaloTag® fusion constructs of human Nup37 or Nup43 (OmicsLink, GeneCopoeia) to isolate the interacting proteins. These proteins were identified using liquid chromatography/ tandem mass spectrometry (LC/MS/MS). Using either HaloTag® fusion protein as bait, we were able to isolate most of the core components of the sub-complex, Nup160, Nup133, Nup107, Nup96, Nup85, Nup43 and Nup37. In addition, we isolated other interacting factors such as nuclear migration protein (NUDC) and TPR nucleoprotein (Figure 4). This isolation of the endogenous counterparts indicates proper incorporation of the HaloTag® fusion proteins within the NPC complex.
Figure 4. Capture of macromolecular complex Nup 107-160.
HEK 293 cells (12 million) were transiently transfected with either HaloTag®-Nup37, HaloTag®-Nup43, or HaloTag® Control Vector. The cells were collected 72 hours post-transfection and processed following the pull-down protocol in the HaloTag® Mammalian Pull-Down and Labeling Systems Technical Manual #TM342. Panel A. Twenty percent of the SDS eluted samples were separated by SDS-PAGE and stained with silver (Bio-Rad). Panel B. The remaining eluted samples were analyzed by LC/MS/MS and the proteins identified.
Summary
The HaloTag® Mammalian Pull-Down and Labeling System provides an efficient and robust purification method for the isolation of in vivo binary or higher order protein complexes from mammalian cells. The rapid, specific and covalent capture of the HaloTag® fusion proteins onto the HaloLink™ Resin yields an optimized protocol that emphasizes speed but is gentle enough to preserve protein complexes with high purity and low background. Recovered protein partners can be analyzed by a variety of downstream applications including mass spectroscopy for the identification of protein partners. Using the HaloTag® TMRDirect™ fluorescent ligand supplied with the system, complementary labeling and live-cell imaging studies can be performed. In addition, the same HaloTag® construct can be used to perform other applications, such as the study of protein:DNA interactions using the HaloCHIP™ System(8)
.