title: Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli.
abstract: Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys.
authors.affiliation: Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.
authors.forename: Alexsandra R M
authors.initials: AR
authors.lastname: Favacho
authors.forename: Eleonora
authors.initials: E
authors.lastname: Kurtenbach
authors.forename: Silvia I
authors.initials: SI
authors.lastname: Sardi
authors.forename: Vera S
authors.initials: VS
authors.lastname: Gouvea
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000276:immunology / Q000821:virology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D016601:RNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D012401:Rotavirus / Q000235:genetics / Q000276:immunology; D012400:Rotavirus Infections / Q000175:diagnosis / Q000276:immunology / Q000662:veterinary; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C079234:NS35 protein, rotavirus