ИСТИНА

Nuclear export protein of influenza virus A (NEP) is involved in a number of important phases in the virus life cycle. NEP is prone to aggregation, while expressed in our earlier designed bacterial expression system. Preventing this undesirable process requires an understanding the basic mechanisms of this protein self-assembly. The objective of this study was to investigate the peculiarities of NEP aggregation and factors affecting this process. To this end, NEP variants with either C- or N-terminal (His)6 -tags (NEP-C and NEP-N) were expressed in E.coli cells, thus obtaining highly purified homogenous solutions after purification on Ni-NTA-agarose column. The formation of oligomers in protein solutions using variable parameters (temperature, pH, concentration, additives, potential disaggregating agents), was analyzed with different techniques: dynamic light scattering (DLS), chemical crosslinking with bifunctional reagent (glutaric aldehyde), gel-filtration chromatography, atomic force microscopy (AFM). DLS measurements have shown the presence of polymeric particles both in NEP-C and NEP-N solutions, with hydrodynamic radius being in the range of 50-500 nm. AFM studies have revealed diverse morphology of the aggregates. Spherical particles (diameter is about 12-14 nm) are mainly presented in NEP-N solution, while fibrillar amyloid-like aggregates (height is 3.5-4.0 nm and length is up to 500 nm) are predominated in case of NEP-C protein. Structures of the N- and Ctermini were proposed to affect the propensity of NEP to aggregation.