comparative production analysis of three phlebovirus nucleoproteins under denaturing or non-denaturing conditions for crystallographic studies比较生产分析的三沙核蛋白质变性或non-denaturing条件下晶体研究.pdfVIP

Comparative Production Analysis of Three Phlebovirus Nucleoproteins under Denaturing or Non-Denaturing Conditions for Crystallographic Studies 1 1 ´ 2 ´ 2 1 1 Violaine Lantez , Karen Dalle , Remi Charrel , Cecile Baronti , Bruno Canard , Bruno Coutard * ´ ´ ´ ´ ´ 1 Architecture et Fonction des Macromolecules Biologiques, UMR 6098 Centre National de la Recherche Scientifique, Universite de la Mediterranee and Universite de ´ ´ ´ Provence, Marseille, France, 2 Unite des Virus Emergents, UMR 190, Aix-Marseille Universite and Institut de Recherche pour le Developpement, Marseille, France Abstract Nucleoproteins (NPs) encapsidate the Phlebovirus genomic (-)RNA. Upon recombinant expression, NPs tend to form heterogeneous oligomers impeding characterization of the encapsidation process through crystallographic studies. To overcome this problem, we set up a standard protocol in which production under both non-denaturing and denaturing/ refolding conditions can be investigated and compared. The protocol was applied for three phlebovirus NPs, allowing an optimized production strategy for each of them. Remarkably, the Rift Valley fever virus NP was purified as a trimer under native conditions and yielded protein crystals whereas the refolded version could be purified as a dimer. Yields of trimeric Toscana virus NP were higher from denaturing than from native condition and lead to crystals. The production of Sandfly Fever Sicilian virus NP failed in