Download Purification of GST::TaABF1 Fusion Protein in Order to Assess its

Purification of GST::TaABF1 fusion protein in order to assess its
phosphorylation by endosperm proteins
Jessica Moore, Russell Johnson
Department of Biology, Colby College, Waterville, ME 04901
Introduction
TaABF1
ABA
Failure to detect phosphorylation of
GST::TaABF1 fusion protein
Methods
glutathione
affinity column
LEA genes (HVA1)
Peptide T111
PKABA1
TaABF1
α-Amylases (Amy32b)
& proteinases
GA
QESFSLPPPCCR
GST::TaABF1
GST::TaABF1
GST::TaABF1
TaABF1:
GST::TaABF1
I
II
III
GST::TaABF1
purified from E. coli
+
endosperm
proteins
b ZIP
N
C
conserved regions
DNA binding
region
 N-terminal conserved regions and DNA binding
region contain serine residues that can possibly be
phosphorylated as a means of regulation
 TaABF1 mRNA & protein levels do not increase in
presence of ABA → TaABF1 most likely regulated
post-translationally
 PKABA1 shown to phosphorylate TaABF1
 TaABF1 has various degrees of phosphorylation in
vivo
Abstract
Transcription factor TaABF1, a member of the ABA
response element binding factor family, has been
shown to have an important role in the signaling
pathways of gibberellin (GA) and abscisic acid
(ABA) in cereal grains. TaABF1 has also been found
to be phosphorylated in vivo in aleurone cells and is
possibly regulated by kinases. In order to investigate
whether TaABF1 can be phosphorylated by kinases
in wheat grains and which regions of the protein are
phosphorylated, the GST::TaABF1 fusion protein
was purified by a glutathione affinity column and a
phosphorylation assay with wheat endosperm
proteins was performed. Then, the possibly
phosphorylated GST::TaABF1 fusion protein was
repurified with approximately 10-15 percent recovery
for analysis with mass spectrometry.
Peptide T317
 detect GST::TaABF1
protein fragments
ESAAR
 do not detect these fragments at masses expected if
they were phosphorylated
 no evidence of phophorylation
Conclusions
phosphorylation
assay
repurified
GST::TaABF1
Ten to fifteen percent recovery of protein
after phosphorylation assay
 GST::TaABF1
successfully
isolated from
E. coli bacteria
culture
 10-15%
BSA
GST::TaABF1
recovered after
endosperm extract
phosphorylation
assay (70 kDa)
Bovine
serum
albumin
standard
Molecular
weight
standard
(kDa)
Purified Purified fusion
protein after
fusion
protein phosphorylation
assay
Future Directions
120
100
80
 We are able to successfully purify GST::TaABF1
fusion protein from bacteria cultures
 GST::TaABF1 fusion protein was successfully
recovered after endosperm phosphorylation assay
 Phosphorylation was not detected on the
GST::TaABF1 protein using mass spectrometry
GST::TaABF1
60
50
40
30
20
Acknowledgements
I would like to thank Russell Johnson for his help in designing and carrying
out this experiment. I would also like to thank Greyson Butler for his help in
carrying out a portion of the experiment.
 perform endosperm protein phosphorylation assay for
longer
 purify larger amounts of GST::TaABF1→ easier to
analyze via mass spectrometry
 collaborate with other labs who have more sensitive
mass spectrometers
 other ways to detect phosphorylation
References
Gómez-Cadenas, A., Verhey, S.D., Holappa, L.D., Shen, Q. Ho, T.-H.D., and Walker-Simmons, M.K.
(1999) “An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene
expression in barley aleurone layers.” Proc Natl Acad Sci USA 96:1767-1772.
Harris, L.J., Martinez, S.A., Keyser, B.R., Dyer, W.E., and Johnson, R.R. (2013) “Functional Analysis
of TaABF1 during abscisic acid and gibberellin signaling in aleurone cells of cereal grains.” Seed Sci
Research, 23-2: 89-98.
Johnson R.R., Wagner, R.L., Verhey, S.D., and Walker-Simmons, M.K. (2002) “The abscisic acidresponsive kinase PKABA1 interacts with a seed-specific abscisic acid response element binding factor,
TaABF, and phosphorylates TaABF peptide sequences.” Plant Physiol 130:837-846.
Johnson, R.R., Shin, M., and Shen, J.Q. (2008) “The wheat PKABA1-interacting factor TaABF1
mediates both abscisic acid-suppressed and abscisic acid-induced gene expression in bombarded
aleurone cells.” Plant Mol Bio 68:93-103.