Download The (Indirect) Costs of Conducting Research: A study of

A Study of Starch Metabolizing Proteins
Pam
1
Brewer-Michael ,
1Marshalltown
Tracie
Myers /James
2
Laboratory
Community School District, 2Iowa State University, Ames, Iowa
ABSTRACT
In order to perform in vitro protein-protein interaction
studies of enzymatic Maize proteins in relation to starch
architecture, working quantities of the proteins of interest
must be available for study. Five Maize proteins were
affinity purified from E.coli cultures transformed with pET
and Gateway vectors containing N-Terminal affinity tags
fused to cDNAs coding full length or specific domains for
SSI, SSII, SSIII, SBEIIa, or SBEIIb proteins.
Verification for affintiy purification of proteins with SDS
PAGE electrophoresis were completed. Tandem (MS-MS)
mass spectroscopy and phosphorylation studies were
performed on the purified proteins.
2
Hennen-Bierwagen ,
RESULTS
Starch: Food and Non-food/Industrial Applications
Food
Bread
Baby food
Low fat foods
Noodles
Snacks
Soups
Sauces/mayonnaise
Meat sausages
Beverage
Plastic
Soft drinks
Beer
Alcohol
Coffee
Biodegradable dinnerware
Golf tees
Star ch g ranules:
~75% A m ylopectin (AP)
~25% A m ylose (A M )
Paper
Corrugated board
Cardboard
Paper
Printing paper
Packaging material
Various
Oil drilling
Foundries
Water treatment
Detergent
Glue
Baby diapers
Hygienic diapers
Stain remover
Confectionary
High-boiled sweets
Jelly gums
Jellies
Marmalade/jam
Ice cream
Dairy cream
Fruit fillings
Proteins of projected molecular weight were present in
each of the five transformed cell cultures. (a) The bound
protein/matrix contains not only proteins of interest but
other proteins as visualized from gel electrophoresis and
Western Blotting procedures. Phosphorylation of SSIII
and SBEIIb proteins were not confirmed through Diamond
ProQ staining. (b) Tandem Mass Spectroscopy of one gel
purified sample was successful.
a
S AB W
b
mp
mp
B
Building
HYPOTHESIS
Under the broader question directed at starch biosynthetic
enzymes physically interacting to form multi-protein
complexes: Useful quantities five Maize proteins can be
produced, isolated, purified, and amino acid sequence
verified from E.coli.
METHODS
Pharmacy
Tablets
Dusting powder
Textiles
Agriculture
Fabrics
Yarns
Energy
Seed coating
Fertilizer
Ethanol feedstock
Concrete
Gypsum board
Plaster
Mineral fibre tiles
BACKGROUND
The study of starch metabolism provides several key
understandings including:
Fundamental plant metabolism
•Fusion constructs of Maize cDNA of SSI, SSII, SSIII,
SBEIIa and SBEIIb in PET or GATEWAY vectors were
electroporolated into E.coli cells.
Developmental regulation
Evolutionary relationships
•Cell pellets from 500 ml IPTG induced cultures were
sonicated and affinity purified with S-agarose or GST
matrices.
Starch is an Important Agricultural Resource and
•Protein eluates from affinity purification were visualized
by SDS PAGE electrophoresis.
Currently all starch based products begin with the same
glucose polymer and all structural modifications occur
during processing. With a better understanding of the
synthesis process including protein enzymes, in vivo
production of novel and more useful starch structures may
be possible in the future.
•Phosphorylation protocols were performed with 4 of the
protein extracts under two different concentration
conditions.
•MS-MS Tandem Mass Spectroscopy was completed on
one of the gel purified protein samples.
has diverse Food and Industrial Applications
A
O
O
HO
O
HO
n on red uc in g
e nd
ACKNOWLEDGEMENTS
Special thanks to Tracie Hennen-Bierwagen,
O
CH2
O
O
O
(1 6 )
b on d
O
O
A c ha in
O
O
 (14 )
b on d
O
O
OH
n on red uc in g
e nd
a
a
a morp ho us
lame lla
c rys talline
lame lla
A p side
c ha in
c lu ste rs
a
a
re du cing
e nd
Dr. Alan Myers, and Dr. Martha James.
DISCUSSION
Proteins of expected molecular size were produced by
transformed E. coli cell cultures and separated by affinity
purification. The s-agarose protocols did not result in
highly pure samples. Phosphorylation did not appear to be
successful in two different concentrations and incubation
times, possibly due to the large amount bound protein
present on matrix. This work suggests that E. coli as a
model organism is capable of manufacturing Maize
proteins of the correct size and sequence for in vitro
study. Phosphorylation is necessary not only as a possible
regulation mechanism but also for correct 3- dimensional
structure (folding). Possible additional studies include
optimizing isolation, phosophorylation, and amino acid
sequencing of the purified proteins..
REFERENCES
Hennen-Bierwagen, Tracie. Identification of Protein/Protein Interaction in Starch Metabolism. Dissertation Research Proposal. November,
2006.
James, Martha. Complex Functional Interactions that Determine Starch Architecture. Presentation at 49th Annual Maize Genetics
Conference, March 2007.
re du cing
e nd
~1 n m
B
~10 nm
B ch ain
S-Filtered Sonicate AB- Eluate after
binding W- Pooled column washes BSBEIIa Protein on matrix
M - no phophorolation treatment
P - phosophorlation treatment
Myers, Alan, Morell, Mattew, James, Martha, and Ball, Steven. Recent Progress toward Understanding Biosynthesis of the
Amylopectin Crystal. Plant Physiology, April 2000.