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

13Citations
Citations of this article
39Readers
Mendeley users who have this article in their library.

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.

References Powered by Scopus

Receptor binding and membrane fusion in virus entry: The influenza hemagglutinin

2375Citations
N/AReaders
Get full text

Activation of influenza A viruses by trypsin treatment

608Citations
N/AReaders
Get full text

Role of hemagglutinin cleavage for the pathogenicity of influenza virus

572Citations
N/AReaders
Get full text

Cited by Powered by Scopus

An avian influenza H5N1 virus vaccine candidate based on the extracellular domain produced in yeast system as subviral particles protects chickens from lethal challenge

24Citations
N/AReaders
Get full text

Development of a universal influenza vaccine using hemagglutinin stem protein produced from Pichia pastoris

23Citations
N/AReaders
Get full text

A prime/boost vaccination with HA DNA and Pichia-produced HA protein elicits a strong humoral response in chickens against H5N1

18Citations
N/AReaders
Get full text

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Cite

CITATION STYLE

APA

Kopera, E., Dwornyk, A., Kosson, P., Florys, K., Saczyńska, V., Debski, J., … Grzelak, K. (2014). Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris. Acta Biochimica Polonica, 61(3), 597–602. https://doi.org/10.18388/abp.2014_1882

Readers' Seniority

Tooltip

PhD / Post grad / Masters / Doc 15

52%

Researcher 10

34%

Lecturer / Post doc 3

10%

Professor / Associate Prof. 1

3%

Readers' Discipline

Tooltip

Biochemistry, Genetics and Molecular Bi... 10

43%

Agricultural and Biological Sciences 8

35%

Chemistry 3

13%

Engineering 2

9%

Save time finding and organizing research with Mendeley

Sign up for free