Production of hepatitis B virus preS polypeptide in Escherichia coli by mutation of the 5'-end coding sequence and its purification and characterization.
The preS1 and preS2 antigens (preS Ag) of hepatitis B virus (HBV) elicit virus-neutralizing and protective antibodies which can overcome nonresponsiveness to the currently available vaccine for HBV and also carry the attachment site to HBV hepatocyte receptor. Therefore, in order to study the development of more effective vaccine and the receptor-ligand interaction, it will be helpful to obtain high-level production of the preS Ag from bacteria. We have found that the native preS region gene was not expressed under the control of commonly used promoters in Escherichia coli. By site-directed mutagenesis of some nucleotides at the 5'-end of the preS1 region gene, we have generated a mutant gene which is highly expressed in soluble form in E. coli. The produced polypeptide could be efficiently purified by 20% ammonium sulfate precipitation and a gel permeation chromatography and the purified polypeptide was demonstrated to exhibit the antigenicity and the immunogenicity of the preS1 and preS2 Ag, suggesting that it is functional.
Kim
H S
HS
Protein Engineering Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST, Yuseong, Taejon, South Korea.
Kim
Y K
YK
Ryu
S E
SE
Hong
H J
HJ
D003001:Cloning, Molecular; D004926:Escherichia coli; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000276:immunology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility
D006514:Hepatitis B Surface Antigens; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C050806:presurface protein 1, hepatitis B surface antigen; C050807:presurface protein 2, hepatitis B surface antigen