Structural characterization of recombinant hepatitis B surface antigen protein by mass spectrometry.
The primary structure of recombinant hepatitis B surface antigen protein produced in yeast has been confirmed by mass spectrometric peptide mapping. These studies corroborate more than 85% of the amino acid sequence derived by sequencing of the gene and identified the presence of an acetyl moiety on approximately 70% of the NH2-terminal methionine residues. Prior to the present work, direct structural analysis was largely prevented by the insolubility of this integral membrane protein and its primary degradation fragments in aqueous buffers and by partial blockage of the NH2 terminus. These difficulties were overcome by preparative isolation using electroelution of the monomeric 226 amino acid protein from a polyacrylamide electrophoretic gel in the presence of sodium dodecyl sulfate. Chymotryptic digestion of the reduced and carboxymethylated monomer produced a large number of small, predominantly hydrophobic peptides ideally suited for peptide mapping by fast atom bombardment mass spectrometry. The percentage of NH2-terminal methionine blocked by acetyl was determined by a new strategy involving cyanogen bromide cleavage, permethylation, and gas chromatography/mass spectrometry identification and quantitation of the N-methyl-N-acetylhomoserine produced.
Hemling
M E
ME
Department of Physical and Structural Chemistry, Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406-0939.
Carr
S A
SA
Capiau
C
C
Petre
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D006514:Hepatitis B Surface Antigens; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011994:Recombinant Proteins
D000596:Amino Acids; D006514:Hepatitis B Surface Antigens; D007202:Indicators and Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002918:Chymotrypsin