Characterization of recombinant human neuron-specific enolase and its application to enzyme immunoassay.
Human gamma-enolase cDNA prepared by reverse transcriptase-polymerase chain reaction was cloned into the Escherichia coli expression vector pKK223-3. The resulting plasmid, pHTK503, expressed human gamma-enolase as a 46-kDa protein in SDS-PAGE, and in the cells as the active gamma gamma form (designated as recombinant human NSE; R-NSE). R-NSE was purified from E. coli by several chromatographic elutions. Finally, 6.0 mg of R-NSE from 8.1 g cells was purified with a specific activity of 86 units/mg protein. The structural properties of R-NSE were compared with the NSE purified from human brain tissue (B-NSE). The biochemical and enzymatic characteristics were essentially the same, except for the isoelectric point (4.5 for B-NSE and 4.7 for R-NSE). In an NSE immunoassay system, R-NSE and standard NSE were almost equal in reactivity to the anti-NSE antibody. These results indicate that R-NSE can be used as standard assay material.
Aoki
T
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Higashi-Nippon-Gakuen University, Hokkaido, Japan.
Kimura
M
M
Kaneta
M
M
Kazama
H
H
Morikawa
J
J
Watabe
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D007124:Immunoenzyme Techniques; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012015:Reference Standards
D011994:Recombinant Proteins; D010751:Phosphopyruvate Hydratase