Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

  • Edyta Kopera Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.;
  • Angela Dwornyk Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.;
  • Piotr Kosson Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland.;
  • Katarzyna Florys Institute of Biotechnology and Antibiotics, Warsaw, Poland.;
  • Violetta Sączyńska Institute of Biotechnology and Antibiotics, Warsaw, Poland.;
  • Janusz Dębski Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.;
  • Violetta Cecuda-Adamczewska Institute of Biotechnology and Antibiotics, Warsaw, Poland.;
  • Bogusław Szewczyk Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdańsk, Poland.;
  • Włodzimierz Zagórski-Ostoja Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.;
  • Krystyna Grzelak Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland.;
DOI: https://doi.org/10.18388/abp.2014_1882

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.
Published
2014-09-12
Issue
Vol. 61 No. 3 (2014): Influenza virus
Section
Articles

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