Expression, Purification And Crystallization Of H5N1 Virus' PAc-PB1n Protein Complex

Avian influenza is an acute infectious disease caused by influenza A virus,the recent emergence of highly pathogenic avian influenza A virus strains with subtype H5N1 pose a global threat to human health. Scientists first discovered that Avian Influenza subunit H5N1 could infect human in Hong Kong of China in 1997.After this,patients infected H5N1 were found in some Southeast Asia countries,and more and more countries or areas were involved including Vietnam,Poland and so on.WHO have already confirmed 329 people were infected and 201 died by 2nd,Oct.2007,including 25 people infected and 16 died in China(http://www.who.it).The highly pathogenic avian influenza has brought awful damage to avian and human.As subunits of H5N1 RNA polymerase,PA,PB1 and PB2 could form a Polymerase-RNA complex required by replication and transcription of virus RNA in host cell.PA can induce a generalized proteolysis of both viral and host proteins,and may also be involved in virus assembly.PB1 could interact with other two subunits in order to form steady protein complex,moreover,it is reported to contain the endonuclease activity to cut RNA 5' cap.In contrast,PB2 play a key role in elongation of virus RNA and add a 5' cap to v-RNA.Today,we have not clear knowledge about how RNA polymerese interact with virus RNA and its mechanism.Here,we set up a method to over-expressed two peptides from Avian influenza A virus subtype H5N1(A/goose/Guangdong/1/96) in Escherichia coli strain BL21 including a PB1 amino terminal short peptide covering residues 1-25 and a protein fragment from PA subunit covering residues 257-716.After intensive purification by Glutathione-Sepharose column and Sephadex G-200,we co-purified these two peptides as a complex to about 95%purity.This protein complex was further applied for protein crystallization.The best crystals were obtained by the vapour diffusion method with 1-1.5M sodium acetate as the precipitant at pH 7.9,which laid a foundation for the analysis of the tri-dimensional structure of H5N1 RNA polymerase subunit complex as well as further understanding its biological function.