Cloning, expression, isolation and characterization of the pre-S domains of hepatitis B surface antigen, devoid of the S protein.
The 'pre-S' parts of the envelope protein of hepatitis B virus (HBV) have been proposed to be involved in the infection of hepatocytes by HBV. In order to facilitate the study of these processes, we have developed an expression system to allow the production and purification of large quantities of the pre-S protein. To obtain a protein containing all of the pre-S sequence and only this sequence, mutations were introduced into the HBV(ayw) genome to create an NdeI restriction site at the initial ATG of the large surface protein gene. Also, stop codons and a BglII restriction site were introduced after the last codon of pre-S2. This fragment was then cloned into the high-expression vector pET-3A. A protein of the expected Mr was expressed at a level of up to 10% of the total soluble protein in HMS174 (DE3) cells, as judged by SDS/PAGE. A rapid purification method has been developed for this protein. The protein retains the polyalbumin-binding activity ascribed to the pre-S2 sequence, and is recognized by both polyclonal and monoclonal antibodies directed against pre-S determinants. Gel filtration chromatography demonstrates that the protein is monomeric and globular, and c.d. spectroscopy indicates that beta-sheet is the major periodic structure.
Delos
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Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298.
Villar
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Hu
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Peterson
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005813:Genes, Synthetic; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000302:isolation & purification; D015151:Immunoblotting; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D014759:Viral Envelope Proteins / Q000235:genetics
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D015345:Oligonucleotide Probes; D011498:Protein Precursors; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C050807:presurface protein 2, hepatitis B surface antigen