TY - JOUR
T1 - Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation
AU - Gai, Zuoqi
AU - Nakamura, Akiyoshi
AU - Tanaka, Yoshikazu
AU - Hirano, Nagisa
AU - Tanaka, Isao
AU - Yao, Min
PY - 2013/11
Y1 - 2013/11
N2 - After crystallization of a certain protein-RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly.
AB - After crystallization of a certain protein-RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly.
KW - DING
KW - HPBP
KW - crystal structure
KW - overexpression
KW - refolding
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U2 - 10.1107/S0909049513020694
DO - 10.1107/S0909049513020694
M3 - Article
C2 - 24121327
AN - SCOPUS:84885601679
VL - 20
SP - 854
EP - 858
JO - Journal of Synchrotron Radiation
JF - Journal of Synchrotron Radiation
SN - 0909-0495
IS - 6
ER -