Characterization of a recombinant hepatitis e protein vaccine candidate by mass spectrometry and sequencing techniques

Abstract

Publisher Summary A baculovirus vector has been constructed that directs the efficient expression of a recombinant 59.1-kDa protein encoded by the Hepatitis E virus ORP-2 region. This protein was purified by chromatographic means and found to be a doublet by Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To evaluate the nature of the 62-kDa protein doublet observed by SDS-PAGE, the purified r62-kDa protein was chromatographed on a Vydac C18 reversed-phased column with the eluting peak evaluated by electrospray mass spectrometry (ES-MS). The r62-kDa protein is resolved into two primary components corresponding to 56.5 and 58.1-kDa, respectively. The predicted molecular mass of the r62-kDa protein using the coding sequence of residues 112 to 660 of the ORP-2 region is 59.1-kDa. These data suggested that a deletion occurred in the molecule, most likely at the amino or carboxyl terminus. Tryptic digestion in conjunction with mass spectrometry and sequence analysis indicated that the amino terminus was acetylated and that the internal sequences were in agreement with the predicted protein sequence. Tryptic peptide analysis revealed as many as 143 peaks by reverse phase high performance liquid chromatography (HPLC). Peaks were selected for LDMS to determine structural integrity and potential post-translational modifications. A peak did not yield a sequence by Edman degradation. However, the molecular mass matched the predicted mass for the amino terminal tryptic peptide taking into consideration the removal of the N-terminal methionine by a cellular aminopeptidase followed by the acylation of the adjacent alanine residue.