구조 연구를 위한 Shigella flexneri 유래 단백질들의 클로닝, 대량발현, 정제

Abstract

Shigella flexneri is a Gram-negative bacterium that causes shigellosis in the terminal ileum and colon. The Shigella genus is composed of four species and each species has many subspecies. Although the bacteria are closely related to human diseases, characterization and structural study are insufficient for Shigella proteins. In this study, as efforts to perform a laboratory scale structural genomics, nine proteins from S. felxneri strain 5a M90T were selected by using UniProt server and XtalPred server. The nine target proteins, including eight hypothetical proteins and one toxin protein, were cloned into the pET21a vector system. Among nine target proteins, eight proteins were overexpressed in E. coli expression system, and four proteins (SF239, SF246, SF682 and SF743) were soluble. These soluble proteins could be successfully purified by using affinity chromatography, ion-exchange chromatography (IEX) and size exclusion chromatography (SEC). In order to determine the structures of purified proteins, X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy were employed. The single crystals of SF246 were obtained and preliminary X-ray diffraction analysis revealed that the crystal diffracted to about 5.0 Å resolutions. In addition, the NMR experiments of SF239 were performed with four different conditions. As a result, we confirmed that SF239 was an intrinsically disordered protein. It is expected that this study could contribute to establish the system for structural genomics.