title: Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.
abstract: The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response.
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Edyta
authors.initials: E
authors.lastname: Kopera
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Angela
authors.initials: A
authors.lastname: Dwornyk
authors.affiliation: Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Kosson
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Florys
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Violetta
authors.initials: V
authors.lastname: Sączyńska
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Janusz
authors.initials: J
authors.lastname: Dębski
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Violetta
authors.initials: V
authors.lastname: Cecuda-Adamczewska
authors.affiliation: Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdańsk, Poland.
authors.forename: Bogusław
authors.initials: B
authors.lastname: Szewczyk
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Włodzimierz
authors.initials: W
authors.lastname: Zagórski-Ostoja
authors.affiliation: Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Krystyna
authors.initials: K
authors.lastname: Grzelak
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D053124:Influenza A Virus, H5N1 Subtype / Q000737:chemistry / Q000235:genetics; D007252:Influenza Vaccines; D051379:Mice; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D011994:Recombinant Proteins