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 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.
journal_name
Plant Mol Bioljournal_title
Plant molecular biologyauthors
Witte CP,Noël LD,Gielbert J,Parker JE,Romeis Tdoi
10.1007/s11103-004-0501-ysubject
Has Abstractpub_date
2004-05-01 00:00:00pages
135-47issue
1eissn
0167-4412issn
1573-5028pii
DO00000501journal_volume
55pub_type
杂志文章abstract:KEY MESSAGE:Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome. Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA...
journal_title:Plant molecular biology
pub_type: 杂志文章
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abstract::There was no discernible effect after incubating recombinant Anabaena Rubisco and carboxyarabinitol 1-phosphate with the product of the Anabaena rca gene. Since the unactivated cyanobacterial Rubisco is not readily inhibited by ribulose 1,5-bisphosphate and fallover is not observed, a genetic basis for the function of...
journal_title:Plant molecular biology
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abstract::The constitutive cytosolic expression of a yeast ( Saccharomyces cerevisiae ) invertase within potato ( Solanum tuberosum ) tubers has previously been documented to produce a dramatic metabolic phenotype in which glycolysis, respiration and amino acid synthesis are markedly enhanced at the cost of starch synthesis. Th...
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abstract::We report the isolation and characterisation of a wound-induced cDNA designated AoPR1 from a suspension of mesophyll cells that had been mechanically isolated from cladodes of light-grown Asparagus officinalis seedlings by grinding in a mortar and pestle. The transcript abundance is up-regulated following cell separat...
journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00026782
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abstract::Micropropagated shoots of three forest tree species, poplar (Populus tremula x P. alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra x J. regia), were inoculated each with six different wild-type Agrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on waln...
journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00040638
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00187572
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00018458
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journal_title:Plant molecular biology
pub_type: 杂志文章,评审
doi:10.1023/a:1026540524990
更新日期:2000-10-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/s11103-006-9092-0
更新日期:2007-02-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
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更新日期:2008-03-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/s11103-020-01049-0
更新日期:2020-11-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1023/a:1005916821224
更新日期:1998-03-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/s11103-018-0812-z
更新日期:2019-02-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
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更新日期:2001-05-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00019191
更新日期:1995-01-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00020195
更新日期:1995-01-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/s11103-006-9099-6
更新日期:2007-02-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00023991
更新日期:1991-03-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00040617
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journal_title:Plant molecular biology
pub_type: 杂志文章
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更新日期:1994-01-01 00:00:00
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pub_type: 杂志文章
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journal_title:Plant molecular biology
pub_type: 杂志文章
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更新日期:1998-07-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
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更新日期:2017-03-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/s11103-013-0075-7
更新日期:2013-07-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00019506
更新日期:1994-12-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00033603
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF00020626
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/s11103-005-3095-0
更新日期:2005-04-01 00:00:00
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journal_title:Plant molecular biology
pub_type: 杂志文章
doi:10.1007/BF01578377
更新日期:1983-07-01 00:00:00
abstract::Using a fruit-specific cDNA as a probe we isolated and sequenced the two corresponding homologous genes (Sn-1 and Sn-2) of the bell pepper (Capsicum annuum) genome. Both genes have a single intron and numerous unusual long inverted repeat sequences. The introns share 87% homology and Sn-2 contains one 450 bp additiona...
journal_title:Plant molecular biology
pub_type: 杂志文章
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