title: Influence of copy number on the expression levels of pandemic influenza hemagglutinin recombinant protein in methylotrophic yeast Pichia pastoris.
abstract: The hemagglutinin (HA) gene of novel Swine Origin Influenza A/California/04/2009 (H1N1) was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA-synthetic gene having α secretory tag under the control of AOX1 promoter was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having single and multiple copy integrants of the expression cassettes were screened for the expression of full length HA protein in the culture supernatant. In order to completely exploit the expression potential of the P. pastoris expression system, a systematic investigation on the influence of gene copy number on the expression of the recombinant protein was made. A panel of Pichia clones carrying increasing copies of the heterologous gene was selected based on Geneticin resistance and SYBR green-based quantitative real-time PCR approach. Using these strategies, recombinant Pichia transformants carrying up to a maximum of four to six copies of the transgene were identified. After optimising the expression conditions for shaker flask culture, the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the expression level of H1N1HA recombinant protein. Our findings clearly suggest that the gene dosage effect play a vital role in high level expression of the pandemic Influenza HA protein in yeast system.
authors.affiliation: Division of Virology, Defence Research and Development Establishment, Ministry of Defence, Govt. of India, Gwalior, MP, India. athmabiotech@gmail.com
authors.forename: T N
authors.initials: TN
authors.lastname: Athmaram
authors.forename: Shweta
authors.initials: S
authors.lastname: Saraswat
authors.forename: Anil Kumar
authors.initials: AK
authors.lastname: Singh
authors.forename: M Kameswara
authors.initials: MK
authors.lastname: Rao
authors.forename: N
authors.initials: N
authors.lastname: Gopalan
authors.forename: V V S
authors.initials: VV
authors.lastname: Suryanarayana
authors.forename: P V L
authors.initials: PV
authors.lastname: Rao
mesh_terms: D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal; D005814:Genes, Viral; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism*; D059765:Homologous Recombination; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: C543345:H1N1 virus hemagglutinin; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins