Abstrakt

Amyloid-like aggregation of human transketolase

Nadejda Davydova

Human transketolase (TKT, EC 2.2.1.1) catalyzes transfers two-carbon units from ketoses (donor) to aldoses (acceptor). Abnormal activity of the enzyme in human tissues (hTKT) is associated with development of some severe pathological processes, including neurodegenerative and oncological diseases. There are some reasons to believe that these effects may be caused by aggregation of hTKT. In particular, the corresponding E.coli TKT is quite prone to aggregation [Jahromi R., 2011]. Crystal structure of hTKT demonstrates high content of ?-structure [Mitschke L. 2010], which is known to be a key element of amyloid fibrils. The objective of this study was to investigate the propensities of hTKT for aggregation and factors affecting this process. Previously hTKT with N-terminal (His)6-tag was expressed in E.coli and isolated from cytoplasm fraction using Ni-agarose affinity chromatography [Meshalkina L., 2013]. However, most of the protein was found in the inclusion bodies (IBs). Preparations of hTKT solubilized from IBs using 1% SDS were highly stable. At the same time, dissolution of IBs in 8 M urea results in formation of SDS-resistant sediment upon incubation for 24 hours at 8