A Study of Starch Metabolizing Proteins Pam Brewer-Michael1 , Tracie Hennen-Bierwagen2, Myers /James Laboratory2 1Marshalltown Community School District, 2Iowa State University, Ames, Iowa

1
A Study of Starch Metabolizing
Proteins Pam Brewer-Michael1 , Tracie
Hennen-Bierwagen2, Myers /James Laboratory2
1Marshalltown Community School District, 2Iowa
State University, Ames, Iowa
RESULTS 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 b mp mp S
AB W B

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.
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.
S-Filtered Sonicate AB- Eluate after binding
W- Pooled column washes B- SBEIIa Protein on
matrix M - no phophorolation treatment P -
phosophorlation treatment

• BACKGROUND
• The study of starch metabolism provides several
  key understandings including
• Fundamental plant metabolism
• Developmental regulation
• Evolutionary relationships
• Starch is an Important Agricultural Resource and
• has diverse Food and Industrial Applications
• 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.

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..

• METHODS
• Fusion constructs of Maize cDNA of SSI, SSII,
  SSIII, SBEIIa and SBEIIb in PET or GATEWAY
  vectors were electroporolated into E.coli cells.
  
• Cell pellets from 500 ml IPTG induced cultures
  were sonicated and affinity purified with
  S-agarose or GST matrices.
• Protein eluates from affinity purification were
  visualized by SDS PAGE electrophoresis.
• 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.

GRAPHIC OR CHART
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. Myers, Alan,
Morell, Mattew, James, Martha, and Ball, Steven.
Recent Progress toward Understanding Biosynthesis
of the Amylopectin Crystal. Plant Physiology,
April 2000.
ACKNOWLEDGEMENTS Special thanks to Tracie
Hennen-Bierwagen, Dr. Alan Myers, and Dr. Martha
James.