Heterologous expression, characterization and evolution prediction of a diaphorase from Geobacillus sp. Y4.1MC1.

ß-hydroxybutyric acid is the most sensitive indicator in ketoacidosis detection, and accounts for nearly 78% of the ketone bodies. Diaphorase is commonly used to detect the ß-hydroxybutyric acid in clinical diagnosis. However, the extraction of diaphorase from animal myocardium is complex and low-yield, which is not convenient for large-scale production. In this study, a diaphorase from Geobacillus sp. Y4.1MC1 was efficiently heterologous expressed and purified in E. coli with a yield of 110 mg/L culture. The optimal temperature and pH of this recombinant diaphorase (rDIA) were 55 °C and 6.5, respectively. It was proved that rDIA was a dual acid- and thermo-stable enzyme, and which showed much more accurate detection of ß-hydroxybutyric acid than the commercial enzyme. Additionally, we also investigated the molecular interaction of rDIA with the substrate, and the conformation transition in different pH values by using homology modeling and molecular dynamics simulation. The results showed that 141-161 domain of rDIA played important role in the structure changes and conformations transmission at different pH values. Moreover, it was predicted that F105W, F105R, and M186R mutants were able to improve the binding affinity of rDIA, and A2Y, P35F, Q36D, N210L, F211Y mutants were benefit for the stability of rDIA.

Highly efficient soluble expression, purification and characterization of recombinant Aß42 from Escherichia coli.

Aggregation of amyloid-ß protein (Aß) is hypothesized to be a seminal neuropathological event in Alzheimer's disease (AD). Recombinant expression and purification of Aß represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. Herein, we report a novel high-yield expression and purification method for Aß42 based on fusion with maltose binding protein (MBP) followed by the soluble polypeptide linker (NANP)3 and a modified tobacco etch virus (TEV) cleavage site before the Aß42. We obtained a final yield of ∼18 mg L-1 of recombinant Aß42 that was confirmed by SDS-PAGE, protein immunoblotting and MALDI-TOF. Finally, thioflavin T fluorescence and atomic force microscopy revealed that the recombinant Aß42 aggregated into long, branched fibrils. Furthermore, the aggregates of the recombinant peptide had a strong cytotoxic effect on PC12 cells. The method described here can therefore be used to efficiently express the soluble fusion protein MBP-Aß42 and obtain high-purity Aß42 peptide, which can be used to understand the molecular mechanism of Aß42 fibrillization and screen new candidate drugs for AD.