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The HaloTag™ and Protein Network Analysis

RNA polymerase I, II, III

Several different tags have been developed and used for affinity purification of protein complexes for the discovery of novel protein interactions (Banks et al. 2012). We have used the Tandem Affinity Purification tag extensively in S. cerevisiae based studies and the FLAG tag in H. sapiens tissue culture based studies. Both of these affinity purification based approaches are in wide use and are well established in the literature. However, the ability to use one affinity tag system for more than just affinity purification would be advantageous to accelerating biological discovery.

We have adopted the HaloTag™ for this reason for ongoing H. sapiens projects in the lab. Derived from a bacterial dehalogenase, the HaloTag™ system allows one to build a construct and cell lines that can be used for both affinity purification of protein complexes and imaging, for example. We first described our use of the HaloTag™ in a collaborative study on the use of the HaloTag™ for the analysis of human RNA Polymerase complexes (Daniels et al., 2012). In this study the POLR2H protein, which is shared between RNA Polymerase I, II, and III, was used as the HaloTag™ bait protein. Remarkably, use of this single subunit pulled down nearly all the major components of these three related, but distinct complexes. In addition, three poorly characterized proteins were also associated with these pull downs and reciprocal HaloTag™ pull downs suggest an association of these proteins with RNA Polymerase complexes. Imaging analysis of HaloTag™ LOC85395, in particular, showed nuclear localization of this subunit and co-localization with POLR2A, a subunit specific to RNA Polymerase II. (Daniels et al., 2012).

Banks CA, Lee ZT, Boanca G, Lakshminarasimhan M, Groppe BD, Wen Z, Hattem GL, Seidel CW, Florens L, Washburn MP. (2014) Controlling for gene expression changes in transcription factor protein networks. Mol Cell Proteomics. Jun;13(6):1510-22. doi: 10.1074/mcp.M113.033902. Epub 2014 Apr 10. Abstract.

Daniels DL, Méndez J, Mosley AL, Ramisetty SR, Murphy N, Benink H, Wood KV, Urh M, Washburn MP. (2012) Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics. J Proteome Res, Feb 3;11(2):564-75. Epub 2012 Jan 3 Abstract

Banks CA, Kong SE, Washburn MP. (2012) Affinity purification of protein complexes for analysis by multidimensional protein identification technology. Protein Expr Purif, Dec;86(2):105-19. Abstract