Expression in Escherichia coli of the cloned polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus.
Cloned cDNA of genomic segment 10 of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was placed downstream from the lambda PL promoter in expression plasmid pRC23 and expressed in Escherichia coli cells. A polypeptide of the same molecular weight (28 kDa) as natural polyhedrin was synthesized at the level of approximately 10% of total host cell protein. This polypeptide was identified as CPV polyhedrin (r-polyhedrin) after comparative studies. The r-polyhedrin did not form any crystalline structure in E. coli cells but instead accumulated in the form of an insoluble inclusion body, even though natural polyhedrin is known to form a crystalline matrix (polyhedra) in infected insect cells. The purified r-polyhedrin complex, like natural polyhedra, was not soluble in neutral or acidic buffer but soluble in alkaline buffer. Upon solubilization, the r-polyhedrin complex did not undergo proteolytic degradation, while natural polyhedra were digested into small peptides by the associated protease. Incubation of r-polyhedrin with natural polyhedra in alkaline buffer, however, degraded the r-polyhedrin, resulting in an identical profile of peptide products to that of natural polyhedra. These results indicate that even though r-polyhedrin molecules produced in E. coli cells are not in the natural conformation, the molecules can present the identical cleavage sites to the polyhedra-associated alkaline protease. Experiments showed that the alkaline protease was associated with the matrix of polyhedra and not with virus particles.
Lavallee
C
C
Department of Molecular Genetics, Nippon Roche Research Center, Kamakura, Japan.
Arella
M
M
Belloncik
S
S
Furuichi
Y
Y
D000468:Alkalies / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000382:microbiology; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics*; D007181:Inclusion Bodies, Viral / Q000648:ultrastructure; D008969:Molecular Sequence Data; D000077183:Occlusion Body Matrix Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis; D012087:Reoviridae / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure; D015678:Viral Structural Proteins / Q000235:genetics*
D000468:Alkalies; D000596:Amino Acids; D000077183:Occlusion Body Matrix Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D010450:Endopeptidases