Production of a Soluble Recombinant Prion Protein Fused to Blue Fluorescent Protein without Refolding or Detergents in Escherichia coli Cells
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概要
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The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 °C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88±0.07 μM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.
- 社団法人 日本農芸化学会の論文
著者
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Arii Yasuhiro
Laboratory Of Molecular Cell Biology Department Of Chemistry And Biological Science College Of Scien
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YAMAGUCHI Hidenori
Laboratory of Molecular Cell Biology, Department of Chemistry and Biological Science, College of Sci
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FUKUOKA Shin-Ichi
Laboratory of Molecular Cell Biology, Department of Chemistry and Biological Science, College of Sci
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