Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope

Abstract

Beyond the rewards of plant genome analysis and gene identification, characterisation of protein activities, post-translational modifications and protein complex composition remains a challenge for plant biologists. Ideally, methods should allow rapid isolation of proteins from plant material achieving a high degree of purity. We tested three purification strategies based on the eight-amino acid StrepII, six-amino acid His 6 and 181-amino acid Tandem Affinity Purification (TAP) affinity tags for enrichment of a membraneanchored protein kinase, NtCDPK2, and a soluble protein, AtSGT1b, from leaf extracts. Transiently expressed StrepII-tagged NtCDPK2 was purified from Nicotiana benthamiana to almost complete homogeneity in less than 60 min and was directly suitable for enzymatic or mass-spectrometric analyses, allowing the identification of in planta phosphorylation sites. In contrast, purification of NtCDPK2 via His 6 tag yielded partially oxidised protein of low purity. AtSGT1b could be isolated after transient expression from N. benthamiana or from transgenic Arabidopsis thaliana as either TAP-tagged or StrepII-tagged protein.While StrepII-tag purification achieved similar yield and high purity as the TAP-tag strategy, it was considerably easier and faster. Using either tagging strategy, a protein was co-purified with AtSGT1b from N. benthaniana and A. thaliana leaf extracts, suggesting that both the StrepII and TAP tags are suitable for purification of protein complexes from plant material. We propose that the StrepII epitope, in particular, may serve as a generally utilizable tag to further our understanding of protein functions, post-translational modifications and interaction dynamics in plants.

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca 2+ affinities

“…NT-SLAC1, CT-SLAC1, and CPK23 cDNAs were cloned in pJET1.2/blunt using CloneJETTMPCR Cloning Kit (Fermentas) and transferred via the introduced restriction sites into destination vectors: In case of CT-SLAC1 and NT-SLAC1 into pXCS-YFP (44) and in case of CPK23 into pXCS-HA-Strep (AY457636) (45) yielding pXCS-NT-SLAC1-YFP, pXCS-CT-SLAC1-YFP, and pXCS-CPK23-HAStrep, respectively. For in vivo pull down experiments see SI Methods.…”

COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation in Arabidopsis

“…The sequences coding for CSN2 and csn2-5 were recombined into pXCSG-HAStrep giving pXCSG-CSN2-HAStrep and pXCSG-csn2-5-HAStrep. To generate pXCSG-HAStrep, the EcoRV gateway rfB cassette was cloned into pXCSHAStrep SmaI site (48).…”

“…StrepII Affinity Purification-StrepII affinity purifications were performed as described (48). For purification of CSN2-HAStrep, 2 g of tissue from 7-day-old seedlings grown on solid MS/10 medium were used.…”

DREB2C Interacts with ABF2, a bZIP Protein Regulating Abscisic Acid-Responsive Gene Expression, and Its Overexpression Affects Abscisic Acid Sensitivity

“…The coding region of DREB2C was fused to EYFP, and the construct was employed to transform Agrobacterium (C58C1). The Agrobacterium was then coinfiltrated with Agrobacterium containing p19 into tobacco leaves (Voinnet et al, 2003;Witte et al, 2004). Observation of the epidermal cells of the infiltrated leaves revealed that yellow fluorescent protein (YFP) signals were localized in the nuclei (Fig.…”

“…Tobacco (Nicotiana benthamiana) leaves were coinfiltrated with the Agrobacterium strains (C58C1) containing the DREB2C-EYFP construct and p19 according to Witte et al (2004). The images of the tobacco epidermal cells were obtained with a fluorescence microscope (Olympus BX51) 30 to 40 h after infiltration.…”