The Avian Influenza Virus A (h5n1) Rna Polymerase Pa Subunit Of The N-terminal Protein Prokaryotic Expression, Purification And Crystal Screening

The influenza A virus RNA-dependent RNA polymerase complex consists of three subunits PB1, PB2 and PA. PB1 is the backbone of the complex, which can bind the 5' and 3' terminal sequences of the viral RNA form the viral promoter and also has endonuclease activity required to snatch capped primers from host pre-mRNAs for viral RNA transcription; PB2 is responsible for recognition and binding the cap structure of host mRNAs; PA is a phosphorylated protein that induces a proteolytic process that decreases its own accumulation levels and those of coexpressed proteins and has a central role in both transcription and replication.We successfully purified the soluble N-terminal of PA to high purity (>95%) though Affinity chromatography and Gel filtration Superdex G-200 chromatography after cloning the gene fragment corresponding N terminal domain of PA (amino acid 10-266) into expression vector pGEX-6p-l and expression in E. coli (BL21). Crystallization screens of PA were initially performed with commercially available reagents (Index , Crystal Screen I and II, Hampton Research) using the hanging-drop vapour-diffusion method . After optimization in pH, PEG of screen I 18 , the rod-like crystals were obtained in 0. 1mol/l MES pH6. 5, PEG8000 25%(w/v), 0. 1mol/l MgCl₂, crystals belong to space group P1 and diffract to 3. 20 A. The unit-cell parameters are a=97A, b=64A, c=57A. Assuming the presence of three molecules in the asymmetric units, the solvent content is estimated to be about 32.5%.