酿酒酵母二腺苷四磷酸磷酸解酶Ⅰ片段的表达、纯化及晶体培养研究

Expression, purification and crystallization of a truncated Saccharomyces cerevisiae diadenosion 5′,5′′′-P1,P4 -tetraphosphate phosphorylaseⅠ

摘要:

摘要：【目的】为了培养用于X-衍射的酿酒酵母二腺苷四磷酸磷酸解酶Ⅰ的蛋白晶体，为研究该酶的三维结构和功能打下基础；【方法】构建了表达酿酒酵母二腺苷四磷酸磷酸解酶Ⅰ片段（Apa1dn16）的表达质粒，用核苷酸序列分析证明了克隆片段的正确性。将重组载体转化大肠杆菌表达菌株BL21（DE3），经IPTG诱导后，用SDS-PAGE检测了蛋白的表达情况。将粗蛋白用Ni-NTA亲和层析分离，收集组分进一步用Superdex 75分子筛纯化。纯化后的蛋白用SDS-PAGE和质谱检验其纯度和正确性，然后用悬滴气相扩散法进行了蛋白结晶条件的初筛。【结果】成功地用大肠杆菌可溶性地高效表达了Apa1dn16蛋白。得到了分子量约为36 kD的单一蛋白条带，纯度在95%以上。质谱结果证明该蛋白纯度高，分子量正确。将纯化后的蛋白用悬滴气相扩散法进行晶体初筛，得到了针状簇晶。此晶体经SDS-PAGE检测，证明为Apa1dn16蛋白晶体。【结论】利用大肠杆菌高效表达体系可以正确表达Apa1dn16蛋白，蛋白经过纯化后，用悬滴气相扩散法可以生长出针状晶体，适合于进一步的三维结构研究。

Abstract:

Abstract: [Objective] To obtain the crystal of 5′,5′′′-P1,P4-tetraphosphate phosphorylaseⅠ(Apa1) of Saccharomyces cerevisiae for X-ray crystal structure and function analysis. [Methods] We amplified the coding region of an N-terminally truncated version of Saccharomyces cerevisiae diadenosion 5′,5′′′-P1,P4-tetraphosphate phosphorylase I (Apa1dn16) , and cloned it into the pET28 derived expression vector. After having screened the recombinant plasmids by PCR and confirmed them by DNA sequencing, we transformed a positive recombinant plasmid into the Escherichia coli BL21(DE3) cells for efficient expression. Then the expression and solubility of the recombinant Apa1dn16 protein were analyzed by SDS-PAGE after proper concentration of IPTG induction. Following that, we collected the soluble Apa1dn16 protein and purified it to homogeneity by sequential Ni-NTA affinity chromatography and Superdex 75 gel filtration, and then detected the purity and molecular weight of the desired protein by SDS-PAGE and mass spectrometry . In addition, we screened the crystallization conditions of Apa1dn16 with Hampton Research kits using the hanging drop vapor diffusion method. [Results] We efficiently expressed an N-terminally truncated Saccharomyces cerevisiae diadenosion 5′,5′′′-P1, P4-tetraphosphate phosphorylaseⅠin Escherichia coli BL21(DE3). The recombinant protein was partially soluble and was purified to homogeneity with a single band of ~36 kDa after SDS-PAGE. Mass spectrometry analysis further confirmed the purity and intactness of the recombinant protein.Moreover, we obtained the needle crystals of Apa1dn16 by hanging drop vapor diffusion method. [Conclusion] Escherichia coli BL21(DE3) is an efficient expression system for producing enough quantity of Apa1dn16 protein. The purified recombinant Apa1dn16 protein is suitable for crystallization and further structural investigation.