Synthesis of hepatitis B virus core antigen polypeptide in E. coli using pKK223-3 plasmid, a vector for expression, with tac promoter.
A hybrid plasmid was constructed by insertion of the HBc gene encoding HBcAg into the pKK223-3 plasmid at the SmaI cleavage site in the correct direction just downstream from the tac promoter and upstream from the rrnB terminator. The recombinant plasmid carrying the HBc gene was introduced into E. coli and cloned. HBcAg was synthesized in E. coli by using the expression plasmid under the regulation of the tac promoter and rrnB terminator. The tac promoter, derived from sequences of trp and lac UV5 promoters, has identical sequences in two domains (-35 and -10 regions) with optimal distance, and the Shine-Dalgarno sequence, which enables protein synthesis to start at the ATG of the adjacent HBc gene. The nucleotide sequence of the HBc gene and its predicted amino acid sequence were almost identical to those previously reported. Purified HBcAg has a molecular weight of 21,500. This polypeptide gave a positive reaction with anti-HBcAg and anti-HBe antibodies, and was assembled into spherical particles 37 nm in diameter. The recombinant plasmid, carrying the HBc gene between the tac promoter (trp-lac hybrid promoter) and the rrnB terminator in expression plasmid pKK223-3, was useful for efficient expression of the HBc gene and production of HBcAg particles in E. coli.
Shirai
M
M
Third Department of Internal Medicine, Kagawa Medical School, Japan.
Watanabe
S
S
Nishioka
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006512:Hepatitis B Core Antigens / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D004269:DNA, Bacterial; D006512:Hepatitis B Core Antigens; D011994:Recombinant Proteins