Purification And Crystallization Of Alanine Dehydrogenase From Bacillus Pseudofirmus

Alanine dehydrogenase （ALD） is an NAD+-dependent amino acid dehydrogenase. It catalyzes the reversible oxidative deamination of L-alanine to pyruvate and ammonia. Alanine dehydrogenase plays an important role in glucose metabolism and amino acid metabolism. As the reaction product, pyruvate is widely used in agriculture, industry and medicine.Alanine dehydrogense from Bacillus pseudofirmus was inserted into the pET-22b（+） plasmid. The recombinant plasmids were transformed into the host strain of E.coli BL21（DE3）, and the ALD protein （NCBI Reference Sequence:WP₀12959278.1） was expressed by inducing with IPTG. Large amounts of ALD protein was initially purified by Ni-NTA affinity chromatography and then the ALD protein was purified by anion exchange chromatography and gel filtration chromatography. Finallly, the high purity of ALD protein was obtained, which was used to protein crystallization.The crystallization conditions of the ALD protein were screened using the vapor diffusion method. The crystallization conditions were optimized by altering the pH, the type and concentration of precipitant, temperature and the concentration of ALD protein. At 289 K, the best condition of crystallization was selected with the hanging-drop method. The crystallization solution consisted of 4%（v/v） Tacsimate pH 5.0 and 8%（w/v） PEG3350, with 9 mmol/L L-proline as the additive. The concentration for crystallization of ALD protein was 10 mg/mL. X-ray diffraction data were collected to 2.87 A resolution. The crystal belonged to space group P212121, with unit-cell parameters a=97.40 A,b=156.73 A, c= 169.10 A, α=90°,β =90°, γ=90°.The crystallization conditions of the binary complex and ternary complex were screened and optimized by the vapor diffusion method. X-ray diffraction results indicated that the resolution of the crystals were poor, so the conditions of crystallization need further optimization. The mutant ALD R15A was expressed and purified, and its crystals were obtained. In order to get some moderately better diffraction data, the crystals of ALD R15A are being optimized.The experiment obtained crystallization conditions of ALD and the diffraction data were analyzed, which laid a foundation for analyzing the three-dimensional structure of ALD and studying its mechanism and biological functions.