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

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.