Abstract
Vacuolar H+-pyrophosphatase (abbreviated V-PPase; EC 3.6.1.1), one of the proton translocation system in the vacuolar membrane, hydrolyzes pyrophosphate and uses the energy for translocating the protons from cytosol into vacuole. The proton translocation causes the pH gradient and membrane potential difference across the vacuole membrane for driving the secondary active transport in the vacuole membrane. V-PPase is a homodimer composed of a single polypeptide with molecular mass of about 80 kD, and there are approximately 16 transmembrane domains in each monomer. The precise protein structure and enzymatic mechanism are still unknown; it would be a quite important breakthrough to solve the high resolution V-PPase structure by X-ray crystallography. In this study, we have overexpressed the mung bean (Vigna radiata) V-PPase in the yeast heterologous expression system and isolated the V-PPase enriched membrane fraction according to the difference of sedimentation coefficient. We have successfully extracted the membrane proteins from membrane fraction by using DDM (n-dodecyl-β-D-maltoside) as the detergent and utilized metal chelate affinity chromatography and size-exclusion chromatography to purify the solubilized V-PPase. Upon purification, the V-PPase still maintains the enzymatic activity of pyrophosphate hydrolysis. Accordingly, we are convinced that these expression and purification protocols were feasible for producing high purity V-PPase proteins with functional conformation. We are currently developing crystallization screening by using vapor diffusion method and the progress for X-ray protein crystallography is promising at this stage.