Abstract 1688 Expression, Purification, and Characterization Attempts of Glucan Phosphatase LSF1

Starch naturally exists as a semi-crystalline, water-insoluble granule structure; thus, its degradation requires several families of enzymes, including glucan kinases, glucan phosphatases, and beta amylases. Reversible starch phosphorylation is critical in diurnal starch degradation in plants and photosynthetic algae. Glucan water dikinase (GWD) preferentially phosphorylates starch at the C3 and C6 positions of the glucose, while phosphoglucan water dikinase (PWD) selectively phosphorylates at the C3 position. This results in the partial solubilization of the starch, which can then be accessed by the beta-amylases 1 and 3 (BAM1 and BAM3) to be degraded into maltose units. The starch degradation process won't be complete without the glucan phosphatase family of enzymes. There are three known members of the plant glucan phosphatase family: Starch Excess4 (SEX4), Like SEX4 1 (LSF1), and Like SEX4 2 (LSF2). We recently reported a direct correlation between the starch dephosphorylation status and the ability to enhance in vitro starch degradation by BAM3. LSF1 has a definitive role in starch metabolism but does not display phosphatase catalytic activity against glucan substrates. Instead, previous research has shown that LSF1 interacts with BAM1 and BAM3 to regulate starch degradation. In this study, we cloned multiple LSF1 constructs in the pET28b vector and tested their solubility and stability under various expression and purification conditions. Our results suggest that full-length LSF1 protein is unstable and insoluble when expressed in and purified from E. Coli. We obtained small quantities of soluble proteins for the DSP-CBM and PDZ-DUF domains. We aim to use these proteins in starch degradation assays and co-crystallization experiments. Our long-term goal is to establish a full-length LSF1 purification strategy to obtain soluble protein in sufficient quantities, so that molecular basis of LSF1 in starch degradation can be investigated using structural and biochemical methods. This study was supported by NSF-MCB-201207