Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope
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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 His6 and 181-amino acid Tandem Affinity Purification (TAP) affinity tags for enrichment of a membrane-anchored protein kinase, NtCDPK2, and a soluble protein, AtSGT1b, from leaf extracts. Transiently expressed StrepII-taggedNtCDPK2 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 His6 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. thalianaleaf 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.
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- Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope
Plant Molecular Biology
Volume 55, Issue 1 , pp 135-147
- Cover Date
- Print ISSN
- Online ISSN
- Kluwer Academic Publishers
- Additional Links
- affinity purification
- chitin-binding domain tag
- HAT tag
- S tag
- StrepII tag
- TAP tag
- Industry Sectors
- Author Affiliations
- 1. Department of Plant-Microbe Interactions, Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné-Weg, 10, Germany
- 2. Laboratoire de Biologie du Développement des plantes, UMR CNRS-CEA-Université méditerranée, 6191, DEVM, CEN Cadarache, France