RESUMO
The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.
Assuntos
Proteínas de Bactérias/química , Proteínas de Fluorescência Verde/química , Corpos de Inclusão/química , Fosfolipases A1/química , Proteínas Recombinantes de Fusão/química , Yersinia pseudotuberculosis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Fosfolipases A1/biossíntese , Fosfolipases A1/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Yersinia pseudotuberculosis/enzimologiaRESUMO
The critically endangered population of Far Eastern leopards ( Panthera pardus orientalis) may number as few as 60 individuals and is at risk from stochastic processes such as infectious disease. During May 2015, a case of canine distemper virus (CDV) was diagnosed in a wild leopard exhibiting severe neurologic disease in the Russian territory of Primorskii Krai. Amplified sequences of the CDV hemagglutinin gene and phosphoprotein gene aligned within the Arctic-like clade of CDV, which includes viruses from elsewhere in Russia, China, Europe, and North America. Histologic examination of cerebral tissue revealed perivascular lymphoid cuffing and demyelination of the white matter consistent with CDV infection. Neutralizing antibodies against CDV were detected in archived serum from two wild Far Eastern leopards sampled during 1993-94, confirming previous exposure in the population. This leopard population is likely too small to maintain circulation of CDV, suggesting that infections arise from spillover from more-abundant domestic or wild carnivore reservoirs. Increasing the population size and establishment of additional populations of leopards would be important steps toward securing the future of this subspecies and reducing the risk posed by future outbreaks of CDV or other infectious diseases.