1. A Study of Starch Metabolizing Proteins Pam Brewer-Michael 1, Tracie Hennen-Bierwagen 2, Myers /James Laboratory 2 1 Marshalltown Community School District, 2 Iowa 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. 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 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. ACKNOWLEDGEMENTS Special thanks to Tracie Hennen-Bierwagen, Dr. Alan Myers, and Dr. Martha James. 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 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.. 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. GRAPHIC OR CHART S-Filtered Sonicate AB- Eluate after binding W- Pooled column washes B- SBEIIa Protein on matrix M - no phophorolation treatment P - phosophorlation treatment