title: Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.
abstract: 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 degrees 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 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.
authors.affiliation: Laboratory of Molecular Cell Biology, Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Japan. arii@chem.aoyama.ac.jp
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Arii
authors.forename: Hidenori
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Fukuoka
mesh_terms: D003902:Detergents / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D018620:PrPSc Proteins / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D003902:Detergents; D005456:Fluorescent Dyes; D018620:PrPSc Proteins; D011993:Recombinant Fusion Proteins; D006493:Heparin