[Expression, purification and crystallization of a truncated Saccharomyces cerevisiae diadenosion 5',5'''-P1,P4 -tetraphosphate phosphorylase I].
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.


CONCLUSIONS
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.
Li
Wenzhe
W
School of Life Science and Technology Tongji University, Shanghai 200092, China. lwz@mail.tongji.edu.cn
Zhang
Junwei
J
Xu
Ningyu
N
Zhou
Congzhao
C
Chen
Yuxing
Y
D003460:Crystallization; D018360:Crystallography, X-Ray; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D005822:Genetic Vectors; D006005:Phosphorylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D015226:Dinucleoside Phosphates; D011994:Recombinant Proteins; C512991:diadenosine-5',5'''-P(1),P(4)-(beta,beta'-methylene)tetraphosphonate; D006005:Phosphorylases