Delftia sp.T3-6酰胺水解酶DamH晶体制备及X-射线衍射分析

Dam H是菌株Delftia sp.T3-6产生的具有酯键水解活性的酰胺酶,对其原核表达、纯化、结晶条件及蛋白晶体X-射线衍射条件进行了研究。经过原核表达,采用Ni-NTA亲和层析法和透析法纯化蛋白,成功获得了适合晶体生长的蛋白Dam H。289 K下采用座滴气相扩散法进行晶体筛选和制备,在蛋白浓度为15 mg/m L及含有0.2 mol/L乙酸铵,pH 7.1,21%(W/V)聚乙二醇3350的缓冲液中获得了理想的蛋白晶体。在低温100 K下通过X-射线衍射仪(Rigaku Micro Max-007 HF)收集了晶体衍射数据。晶体衍射分辨率为3.4,空间群为三斜晶系P1,晶胞参数为a=14.1,b=23.9,c=85.7,α=112.6°,β=77.2°,γ=89.1°。结晶条件的优化和晶体制备为Delftia sp.T3-6 Dam H高分辨率晶体制备提供了参考,晶体衍射数据的收集为Dam H三维结构的解析奠定了前期基础,将有助于阐明Dam H的催化机制。

Crystallization and X-ray Diffraction of an Amide Hydrolase DamH from Delftia sp. T3-6

DamH is a bifunctional hydrolase showing activity to amide and ester bonds.In this study,the heterologous expression, purification, crystallization optimization and X-Ray diffraction of amide hydrolase DamH from Delftia sp. T3-6 was studied.The recombinant DamH was overexpressed in E. coli BL21(DE3) and purified by affinity chromatography of Ni-NTA. Subsequently, crystals were grown with the sitting-drop vapour-diffusion method at 289 K and diffracted at 100 K using an in-house X-ray source.Ideal diffracting crys-tals were obtained using a solution consisting of 0.2 M ammonium acetate,pH7.1,21% ( W/V) polyethylene glycol 3 350 at the protein concentration of 15 mg/mL,and X-ray diffraction data to 3.40 ? resolution were collected in the anorthic space group P1,with unit-cell parameters a=14.1 ?,b=23.9 ?,c=85.7 ?,α=112. 6°,β=77.2°,γ=89.1°.According to the X-ray diffraction data,the three-dimensional structure of DamH from Delftia sp. T3-6 will be elucidated,and it will provide insights into the biochemical properties of DamH.