Exploration of α-Glucan Phosphorylase (Pho1) Interacting
Proteins in Rice Endosperm.
Fabian Botero, Ronni Weekes, Seon-Kap Hwang, Thomas W. Okita
Modeled structure of rice Pho1
Abstract
Starch Phosphorylase 1 (Pho1) is an elongation enzyme that
catalyzes the synthesis of short-chain malto-oligosaccharides in the
early stages of rice endosperm development. Previous studies have
shown that there may be an protein factor present in the developing
rice endosperm that binds to Pho1 to help accomplish this function
(Hikaru Satoh et al, 2008). To this end, Pho1 was purified and
utilized to investigate the presence of proteins in rice endosperm that
exhibit binding interactions with the Pho1 complex. Pho1 was
purified by DEAE-Sepharose FF chromatography, TALONimmobilized metal affinity column (IMAC) chromatography, and
POROS® 20 HQ strong anion exchange media. The purified Pho1
was then bound again onto TALON-IMAC resin and 0.03% H2O2
was then used to immobilize the it by forming a kinetically inert
ternary complex with resin-Co(III). The Pho1 columns were loaded
with soluble proteins from rice endosperm (8 days after flowering) in
the presence of 0 mM, 1 mM ATP, or 4 mM ATP, washed extensively,
and proteins eluted by boiling the resins in 2% -mercaptoethanol
and 2% SDS. SDS-PAGE results showed the presence of an
endosperm protein interacting with Pho1 in the presence of 1 mM
ATP. The protein band was excised from the polyacrylamide gel and
is currently be identified by tandem mass spectrometry.
Background
One of the most abundant plant-derived renewable energy sources are
α-glucans, consisting of starch and malto-oligosaccharides. The
synthesis of these carbohydrates is made possible through the
collaboration of a number of enzymes, including ADP-glucose
pyrophosphorylase, starch synthases, granule-bound starch synthases,
starch
branching
enzymes
and
debranching
enzymes.
Phosphorylases are typically associated with the breakdown of αglucans in budding yeast and animals. While this is largely true for
starch phosphorylase 2, starch phosphorylase 1 (Pho1) has previously
been determined to be found exclusively in developing rice
endosperm and play a significant role in the synthesis of maltooligosaccharides [Hikaru Satoh et al., 2008; Hwang et al., 2010].
Whether or not the enzyme worked alone or as a multi-protein
complex with the assistance of an unknown factor had yet to be
determined. Using a series of anion-exchange and affinity resin
chromatography methods the purification of Pho1 was realized in
order to create a means for harnessing any protein that might interact
with the Pho1 protein from within the rice endosperm matrix by
passing it through a resin with immobilized Pho1 attached to Co(III)
beads.
DEAE-Sepharose FF Chromatography
Induction with 0.1
mM IPTG for 18 h
Harvest
Cell lysis using a
microfluidizer &
centrifugation
(kDa)
170 130 95 72 55 43 34 26 -
DEAE-Sepharose resin is a weak anion-exchanger that binds proteins with
negative surface charges. NaCl was used for elution and the gradient is
shown by the upscale ramp. Fractions 19 – 25, highlighted in red, showed
elution of the desired Pho1 protein (red arrow). M, marker proteins; w.c.,
whole cell extract; Sol., soluble proteins.
TALON-IMAC Chromatography
DEAE-Sepharose FF
chromatography
Flow-through wells show the endosperm proteins that passed through the
column. On the right are the captured protein at various concentrations of
ATP. Larger bands are the Pho1 that was tethered to the beads whereas
the smaller band is the potential interacting protein. Note the unique
polypeptide band (indicated by red arrow) in 1 mM ATP eluate.
Discussion
Upon successful purification of Pho1 the purified protein was covalently
attached to resin-Co(III) via oxidation by 0.03% H2O2 solution. The
results from passing proteins from developing rice endosperm through
this modified Pho1-tethered Co(III) resin clearly show the appearance of
a polypeptide that binds Pho1 in the presence of 1 mM ATP. This
mystery protein band may be the missing factor that Pho1 binds with to
form a multi-protein complex responsible for malto-oligosaccharide
synthesis.
Future Work
TALON-IMAC
chromatography
•LC-tandem mass spectrometry identification of the trapped protein.
•Confirmation of the interaction between Pho1 and the identified protein
in a multi-protein complex.
POROS 20 HQ
Chromatography
Resin-Co(III)-Pho1
tethered filtration of
endosperm proteins
POROS 20 HQ is a strong anion exchange resin that is designed for
greater resolution and faster eluting. The elution peak for Pho1 has been
noticeably sharpened and SDS-PAGE analysis shows elution fractions
with purified Pho1 bands. Upscale ramp depicts salt gradient progression.
SDS-PAGE Analysis of Resin-Co(III)Pho1 Tethered Filtration
Methods
Grow E. coli cells
harboring SH407
POROS 20 HQ Chromatography
Acknowledgements
Special thanks to Dr. Seon-Kap Hwang and Dr. Thomas W. Okita, Ronni Weekes and National Science
Foundation Grant DBI-0605016 for providing funding for this research.
TALON-IMAC resin is an affinity chromatography method that
selectively purifies histidine-tagged proteins. In this case, Pho1 has been
engineered to contain extra six histidine residues on the N-terminus. The
chromatogram shows a sharp peak where the Pho1 was eluted and SDSPAGE analysis shows marked increase in purity.
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