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.

• PMID: 19149172
• PubMed
• PubTator

Reference Type

Journal Article | Research Support, Non-U.S. Gov't

Authors

Li W, Zhang J, Xu N, Zhou C, Chen Y

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Additional Lit For

APA1

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