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.

The DnaJ-Like Zinc-Finger Protein HCF222 Is Required for Thylakoid Membrane Biogenesis in Plants

“…To this end, we used the Gateway-based vector pAUL1 (Lyska et al, 2013a), which confers a 35S promoter and the coding region for a C-terminal 27-amino acid-long triple hemagglutinin (HA) epitope tag to the cDNA. In the T2 progeny of transformed heterozygous plants from both mutants, we were able to identify seedlings that were homozygous for the hcf222-1 locus (five independent transformed plants) or the GK-038B09 T-DNA insertion (four independent transformed plants) but that grew like the wild type under autotrophic conditions (Supplemental Fig.…”

Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles

“…For stable expression in transgenic plants, a genomic fragment of NBR1 containing 2 kb of the predicted promoter and the coding region without the stop codon was amplified (see Table S1 for primer sequences), cloned into pENTR/D-TOPO, and subsequently recombined into pAUL11 (40) to add 3′-end sequences encoding Strep(S)-III and 3xHA tags. The NBR1 fragment containing the 3xHA and SIII extension was reamplified by PCR, cloned into pENTR/D-TOPO, and recombined into pGWB459 or pGWB604 (41) to generate the pNBR1:NBR1-SIII-3xHA-tagRFP and pNBR1:NBR1-SIII-3xHA-GFP constructs, respectively.…”

Higher plant transformation: principles and molecular tools

“…2011). A new series of binary GATEWAY cloning vectors (pAUL1-20) has been generated for C-terminal and N-terminal proteins fused in-frame to four different tags: a single hemagglutinin epitope, a streptavidin-tagII, both epitopes combined to a double tag, and a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site (Lyska et al, 2013). For multiple gene expression, modified GATEWAY cloning systems have been developed (Chung et al, 2005;Kimura et al, 2013;Vemanna et al, 2013).…”