Membrane fusion is an important property of biological systems in general and has been the subject of many investigations. Influenza hemagglutinin (HA) is often used as a model system for these studies. For this reason HA was chosen as a target for a His-Tag introduction by recombinant DNA technologies for use as a step in protein purification. The refinement and simplification of HA purification has potential value, not only in the general study of membrane fusion, but also in the equally important use in different medical applications, such as highly fusogenic liposomes for drug targeting. Six histidine residues were introduced into the HA protein by DNA mutagenesis of the HA gene of A/FPV/Rostock/34, which codes for the wildtype HA protein (HAwt) of 545 amino acids. Amino acids 198-203, situated in the global head region of HA, were changed into histidine residues. This location of a His-Tag within the protein is unusual since it is in contrast to the typical terminal placement of a His-Tag. The genes of HAwt and HA-His were expressed in insect cells (Baculovirussystem), as well as in mammalian cells (Vaccinia-T7-Expressionsystem). In all tests, HA-His was compared to HAwt. Both proteins were characterized using several criteria in order to ensure that the integrity of HA-His was not different to that of HAwt. Neither the glycosylation nor the trimerisation of HAwt and HA-His showed any difference. Receptor binding and induction of membrane fusion for both proteins were also identical. HA-His was purified by using affinity chromatography on a Ni-NTA-Agarose column, which selectively binds proteins that contain stretches of histidine residues. The conditions for this purification were optimized so that HA-His from the starting membranes could be concentrated. The results of this study show that addition of a His-Tag within the exposed head region of the HA protein has no influence on the biochemical or functional characteristics that were examined in this study. In addition, it was shown that purification of a protein by metal ion affinity chromatography is possible even with the untypical internal placement of a His-Tag within the amino acid sequence.
