Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry.
A protein with a molecular mass of approximately 62.10(3), derived from open reading frame 2 (ORF-2) of the hepatitis E virus (HEV: Burma strain), was expressed in a baculovirus expression vector and purified to homogeneity. The recombinant 62 kDa protein appeared to be a doublet, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tryptic digestion in conjunction with laser desorption mass spectrometry (LD-MS) and sequence analysis of the tryptic peptides indicated that the amino terminus was blocked, although no proteolytic degradation occurred. The determined internal sequences of peptides were in agreement with the predicted ORF-2 protein. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) resolved the doublet proteins into two major components with molecular masses of 56548.5 and 58161.4. Confirmation of the amino terminus of the molecule by LD-MS post-ion decay enabled us to tentatively assign the carboxyl terminus of each species at residues 540 and 525. Sequencing of the intact protein by automated carboxyl terminal sequencing confirmed that the carboxyl terminus was truncated and that the sequence assignment predicted by LC-MS was correct.
McAtee
C P
CP
Genelabs Technologies, Inc., Redwood City, CA 94063, USA.
Zhang
Y
Y
Yarbough
P O
PO
Fuerst
T R
TR
Stone
K L
KL
Samander
S
S
Williams
K R
KR
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D016752:Hepatitis E virus / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D018411:Spodoptera; D014614:Vaccines, Synthetic / Q000302:isolation & purification; D014761:Viral Hepatitis Vaccines / Q000235:genetics / Q000302:isolation & purification*; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification*
C103807:62 kDa protein, Hepatitis E virus; D014614:Vaccines, Synthetic; D014761:Viral Hepatitis Vaccines; D014764:Viral Proteins