Expression and Purification of NS1 Protein of Highly Pathogenic Avian Influenza Virus H5N1 in Escherichia coli

Abstract

The highly pathogenic avian influenza (HPAI) is a viral disease of poultry and is a zoonosis. The eighth segment of the viral genomic RNA encodes a nonstructural protein, NS1. NS1 is a viral regulatory protein responsible for virus virulence. This study, produced the 6xHis-tag fusion protein.
The full length NS1 cDNA of A/Chicken/TH/KU14/04 (H5N1) was cloned into an expression vector, pQE80L. The recombinant NS1 was expressed in the E. coli strain DH5α. The results showed that the polyhistidine-tagged NS1 fusion protein was successfully expressed in bacterial cells, but the fusion protein accumulated in the inclusion bodies. The fusion protein was isolated as either a native or denatured form and further purified by precipitation or immobilization on a nickel ion affinity column. Purification  using affinity chromatography under denaturing conditions yielded greater amounts of purer NS1 protein. However, sufficient purity and concentration of the native NS1 protein was obtained after purification using a precipitation method. Both native and denatured, purified NS1 protein reacted specifically with the anti-NS1 chicken serum in a western blot assay. The results indicated that the NS1 protein may be useful for further applications.