Tannase 's Protein Purification : The Cloning, Expression, And Purification Of Tannase Enzyme Obtained From Bacterium L

1501 Words May 4th, 2015 7 Pages
Tannase’s protein purification
The cloning, expression, and purification of tannase enzyme obtained from bacterium L. plantarum were conducted as described (Wu et al., 2013).
Tannase’s protein crystallization, heavy-metal derived crystals preparation and diffraction pattern collection
By using sitting drop vapor diffusion method, the initial apo form of tannase enzyme was crystallized. Then, the initial tannase crystal formed was purified and enlarged by using micro-seeding method (Wu et al., 2013). Both sitting drop and micro-seeding method were performed at 281K (Wu et al., 2013). By either soaking or co-crystalizing the native tannase crystals with heavy metals to generate heavy-metal derived crystals, in order to solve the structure of tannase
For heavy-atom soaking experiment, the native forms of tannase crystals were soaked in the reservoir solution that contained 4.5mM and 6.8mM HgCl2 for 2 days to obtain two mercury-derived crystals.
For co-crystallization method, the tannase protein was co-crystalized with heavy atom compound that were added into the reservoir solution to a final concentration of 25mM for NaBr, 5mM CdCl2, and 15mM CdCl2 using micro-seeding technique to obtain three other heavy-atom derivative crystal.
The original apo form crystal and heavy-atom derived crystals were cryoprotected by using cryoprotectant, which was a mixture of reservoir solution with additional 20% (v/v) of polyethylene glycol 200.
The diffraction data was collected using ADSC…
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