摘要
丙氨酸脱氢酶可逆催化丙氨酸脱氨生成丙酮酸,在氨基酸和酮酸的合成及代谢中至关重要.本研究通过PCR从巨大芽孢杆菌WSH-002中克隆并构建了丙氨酸脱氢酶基因(aldBM066)的原核表达载体,经原核表达后,采用Ni-NTA亲和层析法和阴离子交换色谱法纯化获得蛋白AldBM066,在289 K下以座滴法进行晶体生长条件筛选和制备.通过对蛋白质结晶条件的筛选,最终在蛋白质浓度为15 mg/mL及含有0.1 mol/L乙酸钠(p H 5.0)和2.4 mol/L甲酸钠的缓冲液中获得了理想的蛋白质晶体,晶体大小约为210μm×180μm×150μm,X-射线衍射数据显示,该蛋白质晶体衍射分辨率为2.88,空间群为三方晶系,晶胞参数为a=b=118.71,c=150.51,α=β=90°,γ=120°,每个不对称单位中含有1个AldBM066单体,马修斯系数为2.623/Da,溶剂含量约为53.02%.衍射数据的成功收集为解析巨大芽孢杆菌WSH-002中丙氨酸脱氢酶的三维结构奠定了前期基础,将有助于阐明以单体存在的丙氨酸脱氢酶的催化机制.
Alanine dehydrogenase（ Ald） catalyzes the deamination of alanine to produce pyruvate. Ald plays a pivotal role in the metabolisms of amino acids and ketonic acids. His6 tagged alanine dehydrogenase066（ AldBM066） from Bacillus megaterium WSH-002 was over-expressed in Escherichia coli BL21（ DE3）and purified by Ni-NTA affinity and anion-exchange chromatography. Crystals for X-ray crystallographic analysis were prepared by the sitting-drop vapour-diffusion method at 289 K in a solution of 0. 1 mol/L sodium acetate trihydrate pH 5. 0,2. 4 mol/L sodium format with a protein concentration of 15 mg/mL. The obtained crystal size was approximately 210 μm × 180 μm × 150 μm. The X-ray diffraction data were collected to a resolution beyond 2. 88 in trigonal space group R32,with the unit-cell parameters a = b =118. 71 ,c = 150. 51 ,α = β = 90°,γ = 120°. The asymmetric unit contains one molecule with a calculated Matthews coefficient of 2. 62 3/ Da and a solvent content of 53. 02%. According to the X-ray diffraction data,the three-dimensional structure of AldBM066 from B. megaterium WSH-002 will be readily resolved and will provide insights into the biochemistry of monomeric alanine dehydrogenase.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2015年第10期1071-1076,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
Supported by National Natural Science Foundation of China(No.31300601)
PUMC Youth Fund(No.33320140186)
Natural Science Foundation of Hebei Province(No.C2015205212)
Research Funding of Hebei Normal University(No.L2012Z12)~~
