title: pAUL: a gateway-based vector system for adaptive expression and flexible tagging of proteins in Arabidopsis.
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.
authors: Entwicklungs- und Molekularbiologie der Pflanzen, Heinrich-Heine-Universitaet, Duesseldorf, Germany. dalyska@lbl.gov
authors: Dagmar
authors: D
authors: Lyska
authors: Kerstin
authors: K
authors: Engelmann
authors: Karin
authors: K
authors: Meierhoff
authors: Peter
authors: P
authors: Westhoff
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics; D002736:Chloroplasts / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D039642:Eukaryotic Initiation Factors / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D000939:Epitopes; D039642:Eukaryotic Initiation Factors; C503077:HCF107 protein, Arabidopsis; C115034:HCF136 protein, Arabidopsis; C523214:HCF173 protein, Arabidopsis; D008565:Membrane Proteins; D011506:Proteins