The cleavage of the hemagglutinin protein of H5N2 avian influenza virus in yeast

Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry shell. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) such as H5 or H7 subtype are important pathogens for the poultry industry. Due to genetic reassortments between avian and human influenza viruses, global pandemics may emerge and the naive human immunity could not be ready for them. The full-length HA-encoding gene of H5N2 AIV was inserted into a secretory pPICZαA vector and integrated into the genome of Pichia pastoris by heterologous recombination. The HA protein secretion into the medium was induced with methanol. Besides the expected 69 kDa protein, another smaller fragment about 47 kDa was recognized by an anti-AIV-HA monoclonal antibody in Western blot assay. This is the first report on the cleavage of HA0 into HA1 and HA2 in the methylotrophic yeast P. pastoris. This possibly was due to digestion by proteases from P. pastoris based on the amino acid sequences at the predicted cleavage site, 326R-X-K-R329. With similar modifications to the eukaryotes, large quantity, proper antigenicity, and low cost, this expression system may provide a simple tool to produce HA proteins for further use in preparation of ELISA kits and subunit vaccines.