Expression of human erythrocyte NADH-cytochrome b5 reductase as an alpha-thrombin-cleavable fused protein in Escherichia coli.
Recombinant fused protein containing human erythrocyte NADH-cytochrome b5 reductase (cytochrome b5 reductase, EC 1.6.2.2.) was produced in Escherichia coli, which was linked to the NH2 terminus of beta-galactosidase of the vector pUC13 via a recognition sequence of alpha-thrombin. Cleavage of purified fused protein with alpha-thrombin yielded the enzyme whose apparent molecular weight (32,000) was the same as the native enzyme. The amino-acid sequence from Phe-1 to Leu-10 was determined to be identical to that of the authentic enzyme. The purified enzyme showed an identical absorption spectrum and similar catalytic properties to the native enzyme. Establishment of the expression system would make it possible to determine the reaction mechanism of the enzyme.
Shirabe
K
K
Department of Biochemistry, Medical College of Oita, Japan.
Yubisui
T
T
Takeshita
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003579:Cytochrome Reductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D042966:Cytochrome-B(5) Reductase; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics*; D013057:Spectrum Analysis; D013917:Thrombin / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D001616:beta-Galactosidase; D013917:Thrombin