Effective production of the hepatitis C virus core antigen having high purity in Escherichia coli.
The amino-terminal half of putative nucleocapsid (core) protein (amino acids 1-115) of hepatitis C virus (HCV) was directly overproduced in Escherichia coli under the control of the tac promoter. Overproduction of core antigen was achieved by inserting several target genes and by optimizing the culture conditions, whereas a large amount of directly expressed and purified core antigen has not yet been reported. Although the level of expression was comparable to that of the conventional E. coli fused expression system, our recombinant proteins contain only HCV amino acid sequence. Using recombinant E. coli, overproduced large-scale culture system was achieved in jar-fermenter. A highly purified sample of the expressed protein was obtained by ion-exchange and gel permeation column chromatography in the presence of 8 M urea. From a 3.5 l culture, approximately 440 mg of recombinant core protein was obtained after a two-step purification procedure. An enzyme-linked immunosorbent assay developed using the highly purified antigen satisfactorily diagnosed hepatitis C.
Seki
M
M
Biosciences Laboratory, Research Center, Mitsubishi Kasei Corporation, Yokohama, Japan.
Honda
Y
Y
Kondo
J
J
Fukuda
K
K
Ohta
K
K
Sugimoto
J
J
Yamada
E
E
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics* / Q000276:immunology; D007124:Immunoenzyme Techniques; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics*
D017931:DNA Primers; D011994:Recombinant Proteins; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus