Case Study: H5N1 Hemagglutinin (HA)

Influenza hemagglutinin is a homotrimeric glycoprotein responsible for initiating infection in human cells and is the target of neutralizing antibodies generated by influenza vaccines. It is a complex glycoprotein synthesized as a precursor then processed into HA1 and HA2 subunits and finally assembled as a heterotrimer anchored in the plasma membrane. Avian influenza H5N1 hemagluttinin is expressed on the cell surface of Tetrahymena (Figure 1) is readily purified from Tetrahymena membranes (Figure 2) and retains biological agglutination activity of red blood cells (Figure 3).

Figure 1. HA expression in T. thermophila. Confocal images of T. thermohila expressing the H5 hemagglutinin of H5N1 avian influenza virus and stained with neutralizing mAb 5C5 which recognizes a conformation-dependent antigen on the virus protein (Panel A). Arrows show staining of cilia in overexposed images (Panel B).
Figure 2. Purified full-length H5N1-HA. H5N1 HA was extracted from Tetrahymena membranes, purified by ion exchange chromstogrsphy and resolved by SDS-PAGE. Approximately 125 ug purified HA was recovered from a 1L culture.
Figure 3. H5N1 HA is functionally active. Purified H5N1 HA agglutinates red blood cells. Addition of a conformation-dependent anti-H5N1 HA antibody blocks agglutination of red blood cells.