The identification of protein complexes has been facilitated in recent years by the development of new techniques such as those based on yeast n-hybrid systems. The major limitation of these techniques is that they do not mimic the physiological conditions under which proteins normally function. This can result in the failure to detect certain protein-protein interactions, or worse, to a high false discovery rate. Protein tagging using multiple affinity purification tags is a powerful new tool for the isolation of protein complexes under near physiological conditions. In this approach multiple affinity purification tags is fused in frame to the gene of interest, usually under control of its own promoter, and introduced into the target organism. Stepwise binding to the multiple affinity tags results in isolation of the protein complex containing the tagged protein. The individual components of the mulitprotein complex can then be identified using mass spectrometry (MS). Examples of multiple affinity purification tags include the Tandem Affinity Purification (TAP) tag (Figure 1; Rigaut et al. 1999. Nat. Biotech. 17:1030), and the CHH tag (Honey et al. 2001. Nucl. Acids. Res. 29: e24).
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Figure 1
The tandem-affinity-purification (TAP) tag consists of three components: a calmodulin-binding peptide, a tobacco etch virus (TEV) protease cleavage site and Protein A as an immunoglobulin G (IgG)-binding domain. Cells or organisms are generated that contain TAP-tagged protein(s). Extracts are then prepared under mild conditions and TAP is carried out. The first column consists of IgG beads. TEV protease cleaves the immobilized multiprotein complexes. Another round of binding is carried out on a second column that consists of calmodulin beads. The native complex is then eluted by chelating calcium using EGTA.
Text and figure reproduced from Huber. 2003. Nature Reviews 4: 74 |
In this project we are developing and optimising new and existing multiple affinity purification tags for the standard isolation of multiprotein complexes from higher plants. The analyses will be targeted toward native interactions, using mutant backgrounds in which expression of the endogenous protein is suppressed, and toward ectopically formed complexes, using over-expression lines. Expression of the protein tag in a loss-of-function background can be expected to prevent competition between the introduced and endogenous proteins, thereby facilitating complete complex isolation. However ectopic over-expression in plants is a valuable tool, both for understanding the signalling networks in which proteins function and for altering agriculturally important traits: the development of methods to isolate complexes under these non-native conditions is therefore an important consideration in plant proteomics. We will initially optimise the multiple affinity purification tag method using a few selected target proteins, among which BABY BOOM (BBM). BBM is an embryo-expressed transcription factor that plays a key role in the initiation and maintenance of embryo development in plants (Figure 2; Boutilier et al. 2002. Plant Cell 14:1737).
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Figure 2
35S::BBM over-expression induces somatic embryo formation in Arabidopsis (D, E) and Brassica napus (E). Figure reproduced from Boutilier et al. 2002. Plant Cell 14: 1737. |
Ectopic over-expression of the BBM gene promotes spontaneous embryo development on vegetative tissues of plants and hormone-independent regeneration. Understanding both the native and ectopic BBM signalling pathways will help us optimise the use of this protein for biotechnology applications. Arabidopsis seedlings and in vitro somatic embryos will be used as a model system for isolation of ectopic and native protein complexes, respectively.
Recent poster:
References:
- Boutilier et al. (2002). Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth. Plant Cell 14:1737