NONCODING RNA-PROTEIN COMPLEXES AS TARGETS FOR DEVELOPING NOVEL THERAPEUTIC AGENTS AGAINST DOMESTICATED ANIMAL AND HUMAN PATHOGENS

<b>Project Methods</b><br> Cloning, expression and purification of the proteins. X-ray crystallography requires milligram quantities of high-quality protein. Earlier studies have already demonstrated that the proteins, which bind to ncRNAs investigated in this project, can be readily produced in Escherichia coli. For this project, I will use E. coli BL21(DE3) cells carrying plasmid pMAGIC and containing an appropriate protein-coding gene in vector pMCSG7. Because the expressed proteins have histidine residues (His6-tag) at their N-termini, I will purify them by affinity chromatography. The first chromatography step will produce proteins with purity typically above 80-95%. After desalting, I will carry out the second affinity chromatography. During this step, the His6-tag will be cleaved on the column with a highly specific recombination tobacco etch virus (TEV) protease that cuts only at a designated cleavage site. The second affinity chromatography will produce protein samples with 95-98% purity.In vitro transcription of ncRNAs. In the proposed work, I will use only short protein-binding fragments of ncRNAs. I will synthesize them using the T7 RNA polymerase and chemically synthesized templates.