Expression and characterization of the preS1 peptide of hepatitis B surface antigen in Escherichia coli.
The infectious particles of hepatitis B virus (HBV) contain 3 related surface antigens, i.e., small, medium, and large, all of which are encoded by one large open reading frame with multiple initiation codons. The large surface antigen (L-Ag) contains preS1, preS2, and S regions while both the middle and small surface antigens lack preS1. Several lines of evidence suggested that the preS1 region is involved in the binding of HBV to human hepatocytes as shown by its binding to HepG2 cells and isolated human hepatocyte membranes. To obtain large quantity of preS1 peptide, an expression vector was constructed containing a lac promoter, the 5' half of the beta-galactosidase gene, the Factor Xa tetrapeptide recognition sequence, and the coding region of preS1 plus preS2. This recombinant plasmid constitutively produced high concentration of a fusion protein in inclusion bodies in Escherichia coli. When the fusion protein was treated with Factor Xa, a peptide consisting of the N-terminal 91 amino acids of the preS1 region was released. This preS1 fragment purified by anion exchange chromatography was able to bind specifically to the isolated plasma membranes from human liver. Hence, this recombinant preS1 peptide can be used to identify and isolate hepatocyte receptors for HBV.
Lin
Y
Y
Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
Liu
Y X
YX
Cislo
T
T
Mason
B L
BL
Yu
M Y
MY
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005170:Factor X; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D007181:Inclusion Bodies, Viral / Q000737:chemistry; D008099:Liver / Q000378:metabolism / Q000382:microbiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics*
D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C050806:presurface protein 1, hepatitis B surface antigen; C050807:presurface protein 2, hepatitis B surface antigen; D005170:Factor X