amin tashakor

Firefly luciferase is a light generating enzyme, which is used in different fields of biotechnology and molecular biology. Luciferase has found widespread applications in many areas of genetic analysis such as detecting gene expression, reporter gene assay and proteomics studies such as protein-protein interactions. Despite many advantages, there are some limitations in luciferase-based systems, the most important of which is its low stability. One of the newly developed methods to solve this problem is to take advantage of Deep Eutectic Solvents (DES). One group of DESs is those that composed of organic salts with hydrogen donor, due to which, intermolecular hydrogen bonds cause lower melting point in comparison with each of the component. In this study, we investigated the effects of DES on kinetic properties of wild type and I232R - E354R/Arg356 mutant Lampyris turkestanicus luciferases. For this, both enzymes, wild type and mutant, expressed in BL21, the protein of interest purified through affinity chromatography and used for kinetic studies. Here, we used choline chloride: glycerol as DES. According to the results, the wild type luciferase is much more thermostable in DES than I232R - E354R/Arg356 mutant. Furthermore, the remaining activity of both wild type and mutant luciferases are greater in the presence of DES than those in the absence of DES.

The apoptotic protease-activating factor 1 (Apaf-1) receives the death signal in the intrinsic or mitochondrial pathway of apoptosis. Upon the releasing of cytochrome c from the intermembrane space of mitochondria and binding to Apaf-1 molecules, a heptameric apoptosome complex is formed and triggers the downstream cascade of caspases. Here, for the first time we present spontaneous mutations and recombinations of the Apaf-1 gene and its neighbouring sequences. We sequence 48 colonies containing pcDNA3.1 vector with Nluc/Apaf1 and Cluc/Apaf1 obtained through the quick-change site-directed mutagenesis method, transforming to DH5-α and XL10-Gold at two temperatures, 18 and 37ºC. In 21 of these cases, we found 38 different mutations. Our data suggest that there is a direct relationship between bacterial incubation temperatures and the number of unwanted spontaneous mutations. During our experiment we found that the Apaf-1 gene is much less susceptible to spontaneous mutations when it is transformed into XL10-Gold at 18 ºC. In contrast, a large number of spontaneous mutations were found when the gene of interest transformed into DH5α at 37ºC.