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

• Authors: Edyta Kopera , Angela Dwornyk , Piotr Kosson , Katarzyna Florys , Violetta Sączyńska , Janusz Dębski , Violetta Cecuda-Adamczewska , Bogusław Szewczyk , Włodzimierz Zagórski-Ostoja , Krystyna Grzelak
• Languages of publication: EN

Abstracts

EN

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.

Keywords

EN

Avian influenza; H5N1; recombinant hemagglutinin; Pichia pastoris

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2014
• Volume: 61
• Issue: 3
• Pages: 597-602

Dates

published

2014

received

2014-06-02

revised

2014-07-19

accepted

2014-09-03

(unknown)

2014-09-12

Contributors

author

Edyta Kopera

• Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

author

Angela Dwornyk

• Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

author

Piotr Kosson

• Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland

author

Katarzyna Florys

• Institute of Biotechnology and Antibiotics, Warsaw, Poland

author

Violetta Sączyńska

• Institute of Biotechnology and Antibiotics, Warsaw, Poland

author

Janusz Dębski

• Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

author

Violetta Cecuda-Adamczewska

• Institute of Biotechnology and Antibiotics, Warsaw, Poland

author

Bogusław Szewczyk

• Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdańsk, Poland

author

Włodzimierz Zagórski-Ostoja

• Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

author

Krystyna Grzelak

• Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

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