Avian influenza H5N1 virus, which causes flu in poultry, is a fast pandemic virus and causes major problems for economic and public health. Hemagglutinin (HA) is an essential protein for viral replication because it binds to a receptor on the host cell for viral entry and replication in host cell. Furthermore, hemagglutinin is used to identify the virulence strain of virus. The aim of this research was to clone, express and characterize hemagglutinin from avian flu (H5N1) virus in Pichia pastoris. In this research, the hemagglutinin gene from 3 strains of avian influenza virus, which are two strains of High Pathogenic Avian Influenza (HPAI) and one strain of Low Pathogenic Avian Influenza (LPAI), was ligated into pPICZαA expression vector for expression in P. pastoris KM71. The results showed that all three recombinant HA proteins were expressed in intracellular of yeast, while inducing with 4% (v/v) methanol for 2 days, which was analyzed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. All three recombinant HA proteins were approximately 95 kDa and found as a degraded products which could result from protease activity in yeast cells. All three recombinant HA proteins were partially purified by Diethylaminoethyl cellulose (DEAE) column and were characterized. The result showed that all three partially purified recombinant HA proteins could hemagglutinate red blood cells. Moreover, all of the partially purified recombinant HA proteins were further purified with Sephacryl S-200 column. All three purified recombinant HA proteins were further characterized for hemagglutination activity and Furin cleavage assay. The results showed that all three purified recombinant HA proteins could not hemagglutinate red blood cells and could not be cleaved with Furin enzyme.
