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J Synchrotron Radiat. 2013 Nov;20(Pt 6):854-8. doi: 10.1107/S0909049513020694. Epub 2013 Sep 29.

Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation.

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  • 1Faculty of Advanced Life Sciences, Hokkaido University, Sapporo 060-0810, Japan.

Abstract

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.

KEYWORDS:

DING; HPBP; crystal structure; overexpression; refolding

PMID:
24121327
[PubMed - indexed for MEDLINE]
PMCID:
PMC3795543
Free PMC Article
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