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2014 | 61 | 3 | 597-602
Article title

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

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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.
Year
Volume
61
Issue
3
Pages
597-602
Physical description
Dates
published
2014
received
2014-06-02
revised
2014-07-19
accepted
2014-09-03
(unknown)
2014-09-12
References
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Document Type
Publication order reference
YADDA identifier
bwmeta1.element.bwnjournal-article-abpv61p597kz
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