Protease-like sequence in hepatitis B virus core antigen is not involved in the cleavage processes of core protein in Escherichia coli.
A DNA fragment, coding for hepatitis core antigen (HBcAg), was amplified by polymerase chain reaction and inserted into a lambda PL promoter-derived expression vector. The recombinant plasmid was transformed into Escherichia coli and proteins produced after heat induction were analyzed. In addition to the 21 kDa HBcAg protein, several smaller related polypeptides, particularly one of 17 kDa in size, were also detected with rabbit anti-HBcAg antiserum. Whether the protease-like sequence of core protein involved in the self-cleavage process to form the 17 kDa polypeptide was investigated by a deletion experiment. Our results with a mutant in which 7 amino acids of the conserved protease-like region in the core protein have been deleted suggest that the cleavage does not depend on the presence of these protease-like sequence. In addition, the core protein synthesized from in vitro translation reaction was not cleaved. Core particles from E. coli lysate were purified by sucrose and cesium chloride density gradient centrifugations and subsequently treated with 0.2% of SDS and 0.2% of beta-mecaptoethanol. Immunoblotting analysis, however, did not reveal any conversion of the 21 kDa protein to smaller ones. In conclusion, our results suggest that the protease-like domain at the N-terminus of the core protein does not contain intrinsic autocleavage activity, nor could the HBcAg be converted to smaller antigens by detergent treatment.
Molecular Biology Division, Development Center for Biotechnology, Taipei, Taiwan, ROC.
L H
LH
Hwang
Y J
YJ
Lin
W C
WC
Lai
M S
MS
Lo
D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001483:Base Sequence; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006512:Hepatitis B Core Antigens / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
D006512:Hepatitis B Core Antigens; D014758:Viral Core Proteins; D016282:Aspartic Acid Endopeptidases