Expression and Initial Characterization of the Diverse ADPGlucose Pyrophosphorylase from Oceanbacillus iheyensis

ADPGlucose pyrophosphorylase (ADPG PPase) is the rate-limiting enzyme in starch and glycogen biosynthesis pathways in plants and bacteria, respectively. A detailed understanding of structure-function relationships in this enzyme family will enable the engineering of a highly active and stable enzyme form to increase the supply of biodegradable and renewable carbon. Across species, ADPG PPases exhibit large differences in properties such as stability and allosteric regulation. We focused on ADPG PPase from the bacterium Oceanobacillus iheyensis (Ob.i) due to its extremely halotolerant and alkaliphilic characteristics. Displaying ~30% sequence homology to the well-characterized E. coli and A. tumerfaciens ADPG PPases, this enzyme has unique amino acid residues in conserved regions important for activity and allosteric regulation. We hypothesize that the ADPG PPase from Ob.i will have distinctive kinetic properties that will expand our knowledge of this enzyme family. To test this hypothesis, the enzyme must be expressed in soluble form to evaluate its activity. The E. coli codon optimized Ob.i glgC gene, which codes for ADPG PPase, was subcloned into the pSE420 expression vector and transformed into glgC^- E. coli cells for expression studies. To optimize expression, the following parameters were varied: temperature (15°-37°C), media (LB, TB, ±sorbitol), and inducer concentration (IPTG, 0.1-1mM).  Optimal conditions were found to be in LB media at 30°C, induced with 0.5mM IPTG.  Preliminary assays indicated relatively low enzyme activity. However, substantial amounts of the protein were found in inclusion bodies. We are currently isolating inclusion bodies in efforts to solubilize and renature the recombinant enzyme.