Expression, purification and characterization of Violaxanthin De-epoxidase from Spinach

Plants require sunlight to be able to perform photosynthesis but an excess of intense sunlight is harmful to the organism and therefore needs a method to protect itself. Violaxanthin De-Epoxidase (VDE) is located inside the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to zeaxanthin. Zeaxanthin absorbs the harmful light and dissipates the energy by emitting heat. How this enzymatic reaction is done by VDE is unknown as well as the structure of the full enzyme. The low natural abundance of this protein makes it hard to isolate from its original host and it is therefore overexpressed in Escherichia coli. Isolation and purification of VDE using the soluble proteins fraction resulted in low amounts of VDE with low specific activity but solubilization and refolding of the insoluble protein fraction resulted in high amounts of active VDE. Further purification steps including two-step precipitation and gel-filtration that resulted in monomeric, active VDE (determined to 99 % of protein composition with SDS-PAGE. Verification of expressed protein was made by SDS-PAGE and mass spectroscopy. Protein characterization by Circular Dichcroism (CD), Differential Scanning Fluorimetry (DSF), Dynamic Light Scattering (DLS) and metal analysis was preformed. Crystallization screens JCSG+ and PACT Premier were used to acquire protein crystals but was however not successful. Investigation of enzymatic activity by inhibition with low pH and DTT showed that both these factors can inhibit VDE separately and that the DTT inhibition is reversible