Abstract
Determination of protein function requires tools that allow its detection and/or purification. As generation of specific antibodies often is laborious and insufficient, protein tagging using epitopes that are recognized by commercially available antibodies and matrices appears more promising. Also, proper spatial and temporal expression of tagged proteins is required to prevent falsification of results. We developed a new series of binary Gateway cloning vectors named pAUL1-20 for C- and N-terminal in-frame fusion of proteins to four different tags: a single (i) HA epitope and (ii) Strep-tagIII, (iii) both epitopes combined to a double tag, and (iv) a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site. Expression can be driven by either the 35 S CaMV promoter or, for C-terminal fusions, promoters from genes encoding the chloroplast biogenesis factors HCF107, HCF136, or HCF173. Fusions of the four promoters to the GUS gene showed that endogenous promoter sequences are functional and drive expression more moderately and consistently throughout different transgenic lines when compared to the 35 S CaMV promoter. By testing complementation of mutations affected in chloroplast biogenesis factors HCF107 and HCF208, we found that the effect of different promoters and tags on protein function strongly depends on the protein itself. Single-step and tandem affinity purification of HCF208 via different tags confirmed the integrity of the cloned tags.
Highlights
The majority of cellular processes is accomplished and regulated by proteins
Most of the published Gateway-compatible binary vectors are designed for constitutive expression of transgenes harboring the 35S promoter of cauliflower mosaic virus (CaMV, [5]) or the nopaline synthase (Nos) promoter of Agrobacterium tumefaciens [6]. This strategy may be disadvantageous for purposes like purification of protein complexes via epitope tagged bait proteins because overexpressed proteins might not be associated with their binding partners
Most purification strategies so far rely on the overexpression of the bait protein in wild-type background with the transgenic protein competing for binding partners with the endogenous protein (e.g. [7])
Summary
The majority of cellular processes is accomplished and regulated by proteins. To shed light on the precise function of a protein, tools for detection and/or determination of subcellular localization are required. Gateway-compatible ‘‘pAUL‘‘ vector series for epitope tagging of proteins that are expressed under the control of endogenous Arabidopsis thaliana promoters or the 35 S CaMV promoter [5].
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