Plant Physiology 161: 1903-1917 (2013)

Spatiotemporal seed development analysis provides insight into primary dormancy induction and evolution of the Lepidium DELAY OF GERMINATION1 Genes [W][OA]

Kai Graeber, Antje Voegele, Annette BŁttner-Mainik, Katja Sperber, Klaus Mummenhoff, Gerhard Leubner-Metzger

School of Biological Sciences, Plant Molecular Science and Centre for Systems and Synthetic Biology, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom (KG, AV, GLM);
Web: 'The Seed Biology Place' - www.seedbiology.eu
University of Freiburg, Faculty of Biology, Institute for Biology II, Botany/Plant Physiology, D-79104 Freiburg, Germany (KG, AV, ABM, GLM)
Universität Osnabrück, Fachbereich Biologie, Botanik, D-49069 Osnabrück, Germany (KS, KM)

Received December 24, 2012; Accepted Feburary 19, 2013; Published Feburary 20, 2013.
DOI:10.1104/pp.112.213298

LesaDOG1 protein purification

Supplemental Figure S4. Recombinant GST-LesaDOG1 fusion protein expression and purification.

A, Coomassie stained SDS-PAGE gel showing the IPTG induced expression of the 61 kDa GST-LesaDOG1 fusion protein (arrow) in E. coli. Samples of the growing bacterial culture were taken at indicated timepoints, heated in SDS-loading buffer and directly used in SDS-PAGE.

B, Coomassie stained SDS-PAGE gel showing successive fractions of second round of protein purification process (see methods for details).
1) eluted protein fraction of first purification round after dialysis;
2) matrix bound proteins after incubation of fraction 1 with Glutathion-Sepharose matrix overnight at 4 °C;
3) unbound protein;
4 -7) wash fractions, unbound protein;
8) matrix bound protein after washes;
9) matrix bound protein after TEV cleavage;
10) unbound protein after TEV cleavage;
11 -13) wash fractions, unbound protein;
14) matrix bound protein after washes.
Note: a large amount of GSTLesaDOG1 binds to matrix (2) whilst a small amount is not bound (3) and subsequently washed away (4-7); the matrix bound GST-LesaDOG1 (8) is efficiently proteolytically cleaved at its TEV recognition site resulting in LesaDOG1* appearing at ca. 37 kD and GST* at 25 kD (compare Supplemental Fig. S3) only leaving a small amount of uncut GSTLesaDOG1 (9); relatively pure LesaDOG1* is present in the unbound fractions (10-13) while GST* remains bound to the membrane (14); Fraction 10 was used in western experiments in Figs. 6 and 7 as a control and is referred to as 'DOG1'. Note the remaining presence of GSTLesaDOG1 and free GST* as detected by anti-GST antibody (Fig.6C) in fraction 10, which is hardly visible in the Coomassie stain. M, molecular mass marker.

Article in PDF format (2 MB)
Supplementary data file (4.9 MB)
Abstract

Fig. 1         Fig. 2         Fig. 3         Fig. 4         Fig. 5         Fig. 6         Fig. 7
Fig. S1       Fig. S2       Fig. S3      Fig. S4       Table S1    Table S2
 
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