title: Modification of human neuron-specific enolase for application to radioimmunoassay.
abstract: A recombinant human neuron-specific enolase (R-NSE), isolated from Escherichia coli, could not be used in an RIA system because of instability upon labeling. To apply R-NSE to RIA and to simplify the purification procedure, the N- and C-terminals of R-NSE were modified by tyrosine- and histidine-tagging, respectively. SY-NSE, containing one additional tyrosine residue, was obtained from both soluble and insoluble fractions. More derivatives tagged by two or four tyrosine residues were expressed, but only in the insoluble fraction. SY-NSE and SY-NSE.H6 (containing six histidine residues at C-terminal of SY-NSE) purified from the soluble fraction were applicable to the RIA system, indicating that the addition of a tyrosine residue at the terminal is effective if the antigen is unstable during labeling.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Aoki
authors.forename: M
authors.initials: M
authors.lastname: Kaneta
authors.forename: H
authors.initials: H
authors.lastname: Onagi
authors.forename: J
authors.initials: J
authors.lastname: Morikawa
authors.forename: N
authors.initials: N
authors.lastname: Tsubota
authors.forename: H
authors.initials: H
authors.lastname: Watabe
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000201:enzymology*; D010751:Phosphopyruvate Hydratase / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011863:Radioimmunoassay / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010751:Phosphopyruvate Hydratase