Expression, purification and characterization of neuraminidase from avian flu virus (H5N1) in Pichia pastoris

Abstract

The avian influenza A (H5N1) virus is highly contagious in both wild bird populations and domestic poultry flocks in various continents posing a serious threat to human health. Previous studies revealed that the major surface glycoprotein of the virion namely, neuraminidase (NA) plays an important role in viral replication and infection. The NA is a mushroom-shaped spike protein composed of a single polypeptide chain with three distinct domains: head domain containing the enzyme active site, a highly variable stalk region, and N- terminal signal anchor domain by which the enzyme is embedded in the viral envelope. The function is to hydrolyze the terminal sialic acids of sialoglycans and promotes the release of progeny virus from an infected host cell by destroying receptors on the host cell and the virus itself. The inhibition of NA activity will limit the spread of viral infection thereby suppressing the onset of disease. In this research, the full length and globular head domain of NA were expressed in Pichia pastoris. The induction for the expression was optimal at 30℃ for 5 days in the presence of 4% methanol. From SDS-PAGE and western blot analysis, the sizes of both expressed proteins were approximately 47 kDa. Purification process was carried out for both recombinants. However, only the head domain could be partially purified at 1.28 folds. The kinetic studies revealed the values of K[subscript m] and V[subscript max] at 20.64 ± 2.13 µM and 81.85 ± 2.37 µmol/min/mg protein, respectively. The recombinant activities were competitively inhibited by the antiviral drug, oseltamivir with K[subscript i] and IC₅₀ at 84.15 nM and 30.03 nM respectively. Finally, the effect from the extracts of 10 types of Thai medicinal plants was investigated in comparison. It was found that Vernonia cinerea exhibited nearly complete inhibition at 99.86 ± 0.01% similar to oseltamivir. On the contrary, the lowest inhibition at 25.62 ± 2.21% was obtained from Rhizophora mucronata. The results obtained appear advantageous towards the search for the new antiviral drug for the avian influenza virus H5N1 in the future.