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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.