Open AccessCrystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation
Author(s) -
Gai Zuoqi,
Nakamura Akiyoshi,
Tanaka Yoshikazu,
Hirano Nagisa,
Tanaka Isao,
Yao Min
Publication year - 2013
Publication title -
journal of synchrotron radiation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.172
H-Index - 99
ISSN - 1600-5775
DOI - 10.1107/s0909049513020694
Subject(s) - crystallization , crystallography , escherichia coli , crystal structure , chemistry , crystal (programming language) , mass spectrometry , resolution (logic) , yield (engineering) , protein crystallization , disulfide bond , biophysics , materials science , biology , biochemistry , chromatography , gene , organic chemistry , artificial intelligence , computer science , programming language , metallurgy
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.