Expression, Purification And Crystalization Of Ald And Ald K73A

Alanine dehydrogenase (ALD, EC 1.4.1.1) is an amino acid dehydrogenase that is a NAD-dependent enzyme. It catalyzes a reversible oxidative deamination of L-alanine to pyruvate and ammonia, with NADH as cofactor. It is a key enzyme in regulating amino acid metabolism and glucose metabolism, pyruvate, a promising reaction product, is widely utilized in medicine, pesticides, food and other fields. Based on the protein structure analysis to explore enzyme protein catalytic reaction mechanism of extreme environment, we can provid the conditions for directional transformation enzyme. According to an analysis of crystal structures and catalytic mechanisms of alanine dehydrogenases, a multiple sequence alignment lead to conjecture that Lys73 is also involved in the catalytic reaction.The purpose protein of this experiment is alanine dehydrogenase from alkaliphilic Bacillus pseudofirmus OF4. We constructed a recombinant expression vector successfully and transformd into E.coli BL21 (DE3). A great deal of Aid has been purified by Ni-affinity chromatography and on-exchange column chromatography. Optimize the protein crystallization conditions in order to obtain high-resolution crystals, for example pH and the concentration/type of PEG, temperature and so on. We select the crystallization conditions by some efficient screening kits at 289 K with the hanging drop vapour-diffusion method. The best crystals grew with good reproducibility using a solution containing 0.1 M Tris-HCl pH8.0,0.1 M LiSO4,22%(w/v) PEG 4000,0.05 M NAD+,20% (W/V) Benzamidine hydrochloride 0.22 μL.Using the genome Aid as an emplate, Aid K73A was amplified. The recombinant pET-22b-Ald was introduced into E.coli BL21(DE3) and soluble. The recombinant alanine racemase was efficiently purified to homogeneity by Ni2+ -chelating affinity chromatography and anion exchange chromatography. Optimize the protein crystallization conditions in order to obtain high-resolution crystals, for example pH and the concentration/type of PEG, temperature and so on. We select the crystallization conditions by some efficient screening kits at 289 K with the hanging drop vapour-diffusion method. The best crystals grew with good reproducibility using a solution containing:2 M Ammonium sulfate,25% w/v PEG3350,0.05 M NAD+,0.1 M HEPES, pH7.5. X-ray diffraction data were collected to 4.0 A resolution.Diffraction data of Aid were collected using an in-house X-ray source and an R-AXIS VI++imaging-plate detector at a wavelength of 1.5418 A. X-ray diffraction data were collected to 2.5 A resolution. The crystal belonged to the triclinic space group PI, with unitcell parameters a=87.819 A, b=105.427 A, c=119.62 A, a=87.833°,0=79.031°, y=82.389°. The molecular-replacement method was employed to determine the initial phases using the structure with PDB as a search model. Matthews parameter calculations indicate the presence of 8 to 12 protein molecules in the asymmetric unit.This work determined primarily the crystallization conditions of alanine dehydrogenase Ald and its mutant Ald K73A. And we preliminary analysis the X-ray diffraction data. It will lay the foundation for structure analysis and help us understand the biological function. Moreover, it may support an important guiding for modifying of the deaminase. Finally, it would be used for constructing the gene engineering strain.