Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope.
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 membrane-anchored protein kinase, Nt CDPK2, and a soluble protein, At SGT1b, from leaf extracts. Transiently expressed StrepII-tagged Nt CDPK2 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 Nt CDPK2 via His(6) tag yielded partially oxidised protein of low purity. At SGT1b 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 At SGT1b 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.
Witte
Claus-Peter
CP
Department of Plant-Microbe Interactions, Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, Germany.
Noël
Laurent D
LD
Gielbert
Janine
J
Parker
Jane E
JE
Romeis
Tina
T
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001360:Avidin / Q000737:chemistry; D001483:Base Sequence; D019921:Biotinylation; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D000939:Epitopes; D009842:Oligopeptides; D010940:Plant Proteins; C452192:SGT1b protein, Arabidopsis; D001360:Avidin; D006639:Histidine; D011494:Protein Kinases