重组人细胞红蛋白的表达纯化及谱学表征

Expression,Purification and Spectral Characterization of Recombinant Human Cytoglobin

以可溶性和包涵体两种形式表达纯化得到了重组人细胞红蛋白,并比较了其谱学特征和热稳定性.可溶性蛋白经硫酸铵分级沉淀,再依次经Hiprep 16/10 Q FF阴离子交换柱、Hiload16/60 Superdex75凝胶过滤柱和CM Sepharose FF阳离子交换柱纯化,得到电泳纯的包涵体蛋白;包涵体蛋白经盐酸胍变性溶解、外加血红素重组和柱层析得到了电泳纯的可溶性蛋白.电喷雾质谱表明,以这两种形式得到的蛋白分子量相差153.0,紫外-可见吸收光谱、荧光光谱和圆二色光谱均表明,这两种形式的蛋白在血红素构象上存在差异,其热稳定性也不相同.

Cytoglobin（Cygb） is a recently discovered hemeprotein belonging to the globin superfamily together with hemoglobin, myoglobin and neurogtobin. It is distributed in almost all human tissues. Human cyto- globin is composed of 190 amino acid residues, displays a hexacoordination His-Fe-His in the absence of ex- ternal ligands. In almost all the published literatures, Cygb was expressed in inclusion bodies and purified from this form. Herein, we expressed and purified recombinant human Cygb in soluble form and in inclusion bodies form, and comparely studied their spectral features and thermal stability. The soluble form protein was purified by ammonium sulfate precipitation, Hiprep 16/10 Q FF anion exchange column, Hiload 16/60 superdex 75 size exclusion chromatography and CM Sepharose Fast Flow cation exchange column. The inclusion bodies form protein was purified by dissolving in 6 mol/L guanidinium chloride, renatured with haemin solu- tion and chromatography. ESI-MS results indicate that the molecular weight of the two forms of Cygb is diffe- rence with 153.0. The UV-Vis absorption spectra, fluorescence spectra and circular dichroism spectra show that the heme conformations in the two form cytoglobin proteins are different, and there exists difference be- tween the two forms in their thermal stability.