Expression of the rotavirus SA11 protein VP7 in the simple eukaryote Dictyostelium discoideum.
The outer capsid protein of rotavirus, VP7, is a major neutralization antigen and is considered a necessary component of any subunit vaccine developed against rotavirus infection. For this reason, significant effort has been directed towards producing recombinant VP7 that maintains the antigenic characteristics of the native molecule. Using a relatively new expression system, the simple eukaryote Dictyostelium discoideum, we have cloned the portion of simian rotavirus SA11 genome segment 9, encoding the mature VP7 protein, downstream of a native D. discoideum secretion signal sequence in a high-copy-number extrachromosomal vector. The majority of the recombinant VP7 expressed by transformants was intracellular and was detected by Western immunoblot following gel electrophoresis as two or three bands with an apparent molecular mass of 35.5 to 37.5 kDa. A small amount of VP7 having an apparent molecular mass of 37.5 kDa was secreted. Both the intracellular VP7 and the secreted VP7 were N glycosylated and sensitive to endoglycosidase H digestion. Under nonreducing electrophoresis conditions, over half the intracellular VP7 migrated as a monomer while the remainder migrated with an apparent molecular mass approximately twice that of the monomeric form. In an enzyme-linked immunosorbent assay, intracellular VP7 reacted with both nonneutralizing and neutralizing antibodies. The monoclonal antibody recognition pattern paralleled that found with VP7 expressed in either vaccinia virus or herpes simplex virus type 1 and confirms that D. discoideum-expressed VP7 is able to form the major neutralization domains present on viral VP7. Because D. discoideum cells are easy and cheap to grow, this expression system provides a valuable alternative for the large-scale production of recombinant VP7 protein.
School of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia.
K R
KR
Emslie
J M
JM
Miller
M B
MB
Slade
P R
PR
Dormitzer
H B
HB
Greenberg
K L
KL
Williams
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis* / Q000276:immunology; D036022:Capsid Proteins; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004023:Dictyostelium / Q000235:genetics*; D005822:Genetic Vectors; D006031:Glycosylation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; C050779:VP7 protein, Rotavirus; D014614:Vaccines, Synthetic