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.
Highlights
DING is a family of proteins with molecular mass of approximately 40 kDa, with a conserved N-terminal sequence of DINGGG
The obtained protein was evaluated by X-ray crystallography, and the results indicated that human phosphate-binding protein (HPBP) was correctly refolded to the native structure
It should be noted that the reported HPBP was crystallized as a protein contaminant of paraoxonase 1 purified from human serum (Morales et al, 2006)
Summary
DING is a family of proteins with molecular mass of approximately 40 kDa, with a conserved N-terminal sequence of DINGGG. DING proteins have been identified in a wide range of organisms: prokaryotes, animals, fungi and plants (Berna et al, 2008) They have attracted attention due to recent reports that they are involved in many diseases, including rheumatoid arthritis, lithiasis, atherosclerosis, some tumours, tumour-associated cachexia and bacterial and viral infections (Berna et al, 2009; Bookland et al, 2011; Lesner et al, 2009). It should be noted that the first crystal of HPBP was accidentally grown from a paraoxonase 1 solution purified from plasma (Morales et al, 2006) From this serendipitous discovery, the authors determined the amino acid sequence by combining the X-ray structure and mass spectrometry (Diemer et al, 2008). This is the first report of a system for overproduction of HPBP as well as refolding of DING proteins
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