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

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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
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The diffraction data obtained was processed using XDS program to provide information about the crystal lattice and the symmetry of the protein crystal (Kabsch, 2010). The heavy-atom derived crystals were considered to be insomorphous to the native tannase crystal by showing P1 space group with an average unit cell dimesion of a=24.74, b=62.44, c=59.56, α =90.0o, β=90.45o, γ =90.0o, which indicate triclinic space symmetry.
The obtained hkl intensities were input into CCP4 program for intensity reduction, scaling and merging process (Winn et al., 2011).

Tannase structure determination and refinement
The tannase structure was solved by MIRAS (multiple isomorphous replacement anomalous scattering) using PHENIX software suite (Adams et al., 2010). The initial phases were calculated using five heavy-atom derivative crystals data sets and obtained the figure of merit (FOM) of 0.37 at resolution of 3.5, which acted as the quality control for the phase estimated. Then, the initial phases with weak FOM were refined by electron density and phase modification. Then, an initial model of tannase structure was generated using the automated model building protocol. The initial model consist 89% of the residue in the misaligned unit built in the tannase structure. The initial model produced was first inspected and adjusted manually using COOT
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