Rapid one-step protein puriﬁcation from plant material using
the eight-amino acid StrepII epitope
Claus-Peter Witte*, Laurent D. Noe
, Janine Gielbert, Jane E. Parker and Tina Romeis
Department of Plant-Microbe Interactions, Max-Planck-Institute for Plant Breeding Research, Carl-von-
-Weg 10, 50829 Cologne, Germany (*author for correspondence; e-mail firstname.lastname@example.org)
Present address: Laboratoire de Biologie du De
veloppement des plantes, UMR CNRS-CEA-Universite
e 6191, DEVM, CEN Cadarache, 13108 Saint Paul lez Durance Cedex, France
Received 26 March 2004; accepted in revised form 2 June 2004
Key words: aﬃnity puriﬁcation, chitin-binding domain tag, HAT tag, S tag, StrepII tag, TAP tag
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
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-tagged NtCDPK2 was puriﬁed from Nicotiana benthamiana to almost complete homo-
geneity 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
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.
Abbreviations: AEBSF, 4-(2-aminoethyl)benzenesulfonyl ﬂuoride hydrochloride; AP, alkaline phosphatase;
CDPK, calcium dependent protein kinase; HA, haemaglutinin; IMAC, immobilised metal aﬃnity chro-
matography; Q-TOF MS, quadrupole time-of-ﬂight mass spectrometry; TAP tag, tandem aﬃnity puriﬁ-
An increasing interest in analysing the biochemi-
cal functions, post-translational modiﬁcations and
interacting partners of plant proteins demands
improved puriﬁcation techniques. Efraim Rack-
er’s motto ‘‘Don’t waste clean thinking on dirty
enzymes’’ adopted by Arthur Kornberg in his
‘‘ten commandments’’ (Kornberg, 2003) may
serve as a guide. But how do we best get ‘‘dirty’’
proteins clean? This is greatly facilitated by the
The ﬁrst two authors contributed equally to this work.
Plant Molecular Biology 55: 135–147, 2004.
Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.