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

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Kopera E., Dwornyk A., Kosson P., Florys K., Sączyńska V., Dębski J., Cecuda-Adamczewska V., Szewczyk B., Zagórski-Ostoja W., Grzelak K.

2014

vol. 61

issue 3

597-602

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.

Plant expression systems for production of hemagglutinin as a vaccine against influenza virus

88%

Redkiewicz P., Sirko A., Kamel K., Góra-Sochacka A.

2014

vol. 61

issue 3

551-560

Many examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity.

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2 Acta Biochimica Polonica

2 2014

1 Cecuda-Adamczewska V.

1 Dwornyk A.

1 Dębski J.

1 Florys K.

1 Grzelak K.

1 Góra-Sochacka A.

1 Kamel K.

1 Kopera E.

1 Kosson P.

1 Redkiewicz P.

1 Sirko A.

1 Szewczyk B.

1 Sączyńska V.

1 Zagórski-Ostoja W.

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

Plant expression systems for production of hemagglutinin as a vaccine against influenza virus