Hepatitis B virus envelope L protein particles. Synthesis and assembly in Saccharomyces cerevisiae, purification and characterization.
The hepatitis B virus envelope gene encodes three transmembrane proteins in frame; S, the product of S gene; M, the product of M (pre-S2 + S) gene; and L, the product of L (pre-S1 + pre-S2 + S) gene. Unlike the S and M proteins, attempts to efficiently synthesize L proteins and assemble them into L protein particles in various eukaryotic cells have been unsuccessful, probably because of the presence of the pre-S1 peptide with an unknown function which appears to be inhibitory to the host secretory apparatus. To investigate the role of the pre-S1 peptide, we constructed an L gene fused with a synthetic gene for chicken-lysozyme signal peptide (C-SIG) at the 5'-terminal and placed the resultant gene under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. After the fused-C-SIG peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular mass of approximately 52 kDa at a level of 42% of the total soluble protein. Electron micrographic observation showed that the gene products assembled into 23-nm spherical and filamentous particles. The pre-S peptide of the gene product was deposited into the endoplasmic reticulum (ER) lumen and well-glycosylated. It seemed that the gene products were accumulated as particles in certain specific membrane structures of the yeast secretory apparatus. Moreover, both the amount of mRNAs specific for the L gene and the in vivo stability of the synthesized L proteins did not change significantly by the addition of the C-SIG gene. These findings indicated that, if the pre-S1 peptide penetrates the ER membrane efficiently, the L proteins can be synthesized cotranslationally, translocate across the ER membrane with its S region, and then assemble by themselves into the particle form. Therefore, the pre-S1 peptide may involve weak or reduced signal peptide activity for recognition by the secretory apparatus and/or for the transport of the pre-S peptide into the ER lumen.
Kuroda
S
S
Biotechnology Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Otaka
S
S
Miyazaki
T
T
Nakao
M
M
Fujisawa
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002499:Centrifugation, Density Gradient; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods; D005814:Genes, Viral; D005822:Genetic Vectors; D006515:Hepatitis B virus / Q000235:genetics* / Q000648:ultrastructure; D008715:Methionine / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D021382:Protein Sorting Signals / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000235:genetics*
D021382:Protein Sorting Signals; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; D011061:Poly A; D012313:RNA; D008715:Methionine; D009113:Muramidase