title: Heterologous expression of Bluetongue VP2 viral protein fragment in Pichia pastoris.
abstract: The partial VP2-encoding gene of Bluetongue virus serotype 23 (BTV-23) was amplified using reverse transcription polymerase chain reaction (RT-PCR) and inserted into pPICK9K vector. Recombinant plasmid DNA was integrated into the genome of Pichia pastoris by electroporation and expressed protein was identified by SDS-PAGE. High-level secreted expression was achieved after selecting the Mut ( + ) phenotype with multi-copy integrant in the recombinant yeast. The partial fragment of Bluetongue VP2 protein (BTV VP2) of approximately 45 KDa was secreted into the culture supernatant by the recombinant yeast when induced with methanol. Western and immuno dot-blotting methods confirmed the expressed BTV VP2 protein. The expressed protein has been demonstrated to be immunogenic in rabbits. A standardized method has been evolved for optimal expression and high-level production of the recombinant protein (284 mg/L). This is the first report demonstrating the possibility of mass production of BTV VP2 protein using P. pastoris.
authors.affiliation: Department of Microbiology and Biotechnology, Bangalore University, Jnanabharathi, Bangalore 560056, India.
authors.forename: T N
authors.initials: TN
authors.lastname: Athmaram
authors.forename: Geetha
authors.initials: G
authors.lastname: Bali
authors.forename: Goon G
authors.initials: GG
authors.lastname: Kahng
authors.forename: Sulatha
authors.initials: S
authors.lastname: Dwarakanath
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D001819:Bluetongue / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D001820:Bluetongue virus / Q000235:genetics*; D036022:Capsid Proteins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D005822:Genetic Vectors; D008297:Male; D010446:Peptide Fragments / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D010843:Pichia / Q000235:genetics*; D011817:Rabbits; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014765:Viral Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D010446:Peptide Fragments; C442520:VP2 protein, Bluetongue virus; D014614:Vaccines, Synthetic; D014765:Viral Vaccines