Characterization by mass spectrometry of a recombinant hepatitis delta virus antigen and its proteolytic products.
The recombinant hepatitis delta virus antigen was obtained as a chimaeric protein fused to the C-terminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i.e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of delta antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis delta antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35-40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis delta virus) antisera.
Tecce
M F
MF
Sclavo Research Center, Siena, Italy.
Petracca
R
R
Giuliani
M M
MM
Ruoppolo
M
M
Marino
G
G
Malorni
A
A
Pucci
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000032:analysis*; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D003698:Hepatitis Delta Virus / Q000276:immunology*; D035923:Hepatitis delta Antigens; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011817:Rabbits; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000032:analysis; D016339:Spectrometry, Mass, Fast Atom Bombardment
D000956:Antigens, Viral; D004279:DNA, Viral; D035923:Hepatitis delta Antigens; D011993:Recombinant Fusion Proteins; C442244:hepatitis delta virus large antigen