Effects of Altering Purification Steps on the Enzymatic Activity of Glycerol Phosphate Dehydrogenase When Incorporated in a Recombinant Protein Vesicle

Synthetic vesicles can be used for a variety of applications, ranging from serving as drug delivery vehicles to biosensing capability to studying nanoscale phenomena. In recent years, protein vesicles have showed advantageous qualities, namely increasing biocompatibility and specific bioactivities designed by protein components. In this work, we designed a protein vesicle through self-assembly by combining a recombinant fusion protein, glycerol phosphate dehydrogenase fused with a glutamic acid-rich leucine zipper (GPDH-Z_E), with a complimentary arginine-rich leucine zipper fused to an elastin-like polypeptide (Z_R-ELP). We hypothesized that GPDH-Z_E on the protein vesicle membranes will facilitate an enzyme cascade reaction of glycerol to dihydroxyacetone phosphate (DHAP), which is instrumental in lipid metabolism in the body. The GPDH enzyme provides an active site for glycerol-3-phosphate (G-3-P) to convert to DHAP, which is one of the reactions involved in the enzyme cascade. The GPDH in its commercially used state has successfully converted G-3-P to DHAP, however the enzymatic activity of this enzyme on the protein vesicle membrane of GPDH-ZE has not been sufficient to carry out the reaction in previous trials. My research aims to identify how altering purification steps in the expression of the GPDH fusion protein may affect the enzymatic activity of the GPDH-ZE vesicles. The research will involve incorporating different buffer solutions, different concentrations of those buffer solutions, and the subsequent measure of the GPDH vesicle’s enzymatic activity after these changes have been applied.