[截短的酿酒酵母二腺苷 5′,5″′-P1,P4 -四磷酸磷酸化酶 I 的表达、纯化及结晶]

Li Wenzhe, Zhang Junwei, Xu Ningyu, Zhou Congzhao, Chen Yuxing

School of Life Science and Technology Tongji University, Shanghai 200092, China.

Wei Sheng Wu Xue Bao. 2008 Nov;48(11):1537-42.

OBJECTIVE

To obtain the crystal of 5',5'''-P1,P4-tetraphosphate phosphorylase I (Apal) 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 (Apaldnl6) , 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 Apaldnl6 protein were analyzed by SDS-PAGE after proper concentration of IPTG induction. Following that, we collected the soluble Apaldnl6 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 Apaldnl6 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 phosphorylase I in Escherichia coli BL21(DE3). The recombinant protein was partially soluble and was purified to homogeneity with a single band of approximately 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 Apaldnl6 by hanging drop vapor diffusion method.

CONCLUSION

Escherichia coli BL21(DE3) is an efficient expression system for producing enough quantity of Apaldnl6 protein. The purified recombinant Apaldnl6 protein is suitable for crystallization and further structural investigation.

目的

获得酿酒酵母5',5'''-P1,P4-四磷酸磷酸化酶I（Apal）的晶体，用于X射线晶体结构和功能分析。

方法

我们扩增了酿酒酵母二腺苷5',5'''-P1,P4-四磷酸磷酸化酶I（Apaldnl6）N端截短版本的编码区，并将其克隆到pET28衍生的表达载体中。通过PCR筛选重组质粒并经DNA测序确认后，将阳性重组质粒转化到大肠杆菌BL21(DE3)细胞中进行高效表达。然后在适当浓度的IPTG诱导后，通过SDS-PAGE分析重组Apaldnl6蛋白的表达和溶解性。随后，收集可溶性Apaldnl6蛋白，通过连续的Ni-NTA亲和层析和Superdex 75凝胶过滤将其纯化至均一，然后通过SDS-PAGE和质谱检测所需蛋白的纯度和分子量。此外，我们使用Hampton Research试剂盒通过悬滴气相扩散法筛选Apaldnl6的结晶条件。

结果

我们在大肠杆菌BL21(DE3)中高效表达了N端截短的酿酒酵母二腺苷5',5'''-P1,P4-四磷酸磷酸化酶I。重组蛋白部分可溶，经SDS-PAGE后纯化至均一，呈现一条约36 kDa的单带。质谱分析进一步证实了重组蛋白的纯度和完整性。此外，我们通过悬滴气相扩散法获得了Apaldnl6的针状晶体。