Optimization of solvent media to solubilize TEV protease using response surface method.

BACKGROUND AND PURPOSE: Tobacco etch virus (TEV) protease, a 27 KDa protein, consists of the catalytic domain of nuclear inclusion a (NIa) protein produced by Tobacco etch virus. Because of its unique sequence, TEV protease is used for purging fusion tags from proteins. It also has many advantages such as stability and activity in a board range of temperature and pH and overproduction in Escherichia coli and these benefits make this protease valuable. Despite all these benefits, TEV protease has problems like low solubility (less than 1 mg/mL). There are methods for enhancing protein solubility and in this study, the effect of additives during cell lysis was studied. EXPERIMENTAL APPROACH: Eleven different additives that made twelve different lysis buffers were used and their effect on TEV protease solubility analyzed by Plackett-Burman and response surface methodology methods. FINDINGS / RESULTS: Three best effective additives on TEV solubility (L-proline, sodium selenite, and CuCl2) were selected according to software analysis and the best concentration of them was applied to optimize TEV protease solubility. CONCLUSION AND IMPLICATIONS: The obtained results provided the composition of an optimum solvent for obtaining soluble TEV protease.

Therapeutic Effects of Gallic Acid in Regulating Senescence and Diabetes; an In Vitro Study.

Gallic acid (GA), a plant-derived ubiquitous secondary polyphenol metabolite, can be a useful dietary supplement. This in vitro study's primary purpose was to assess the anti-aging properties of GA using rat embryonic fibroblast (REF) cells, antidiabetic effects via pancreatic islet cells, and finally, elucidating the molecular mechanisms of this natural compound. REF and islet cells were isolated from fetuses and pancreas of rats, respectively. Then, several senescence-associated molecular and biochemical parameters, along with antidiabetic markers, were investigated. GA caused a significant decrease in the ß-galactosidase activity and reduced inflammatory cytokines and oxidative stress markers in REF cells. GA reduced the G0/G1 phase in senescent REF cells that led cells to G2/M. Besides, GA improved the function of the ß cells. Flow cytometry and spectrophotometric analysis showed that it reduces apoptosis via inhibiting caspase-9 activity. Taken together, based on the present findings, this polyphenol metabolite at low doses regulates different pathways of senescence and diabetes through its antioxidative stress potential and modulation of mitochondrial complexes activities.

Rational design of a new mutant of tobacco etch virus protease in order to increase the in vitro solubility.

BACKGROUND AND PURPOSE: Tobacco etch virus (TEV) protease is a protease with high sequence specificity which is useful for the cleavage of fusion proteins. A major limitation of this enzyme is its relatively poor solubility. This study aimed to investigate the effects of some suggested mutations by online tools and molecular dynamics simulation to improve the solubility of TEV protease in vitro. EXPERIMENTAL APPROACH: We designed a rational multi-stage process to determine the solubilizing mutations of TEV protease. At the first stage, all the possible mutations were predicted using online tools such as PoPMuSiC and Eris servers, in which five mutations include N23F, N23L, Q74L, Q74V, and Q74I were suggested for further studies. In the next step, the three dimensional structure of the wild type (WT) and the best mutations were subjected to molecular dynamic simulations to evaluate the dynamic behaviour of the obtained structures. The selected mutation was introduced into the structure using site-directed mutagenesis and expressed in Escherichia coli BL21DE3. After purification, solubility and activity of the purified mutant and WT-TEV proteases were assayed. FINDINGS /RESULTS: By considering the analysis of various factors such as structural and solubility properties, one mutant, N23F, was selected for in vitro studies which led to a 1.5 times increase in the solubility compared to the WT while its activity was decreased somewhat. CONCLUSION AND IMPLICATIONS: We propose N23F mutation, according to computational and experimental analyses for TEV proteases which resulted in a 150% increase in solubility compared to the WT.

Efficient Media for High Lipase Production: One Variable at a Time Approach.

BACKGROUND: Lipase enzymes have applications in a wide range of industries. A crucial determining factor of industrial prices of these enzymes is the culture media composition that is constantly under review by researchers. In this work, for maximum lipase production by Bacillus sp. ZR-5, culture media compositions were optimized using ″one variable at a time″ strategy. METHODS: For this purpose, the culture medium parameters such as low and high cost carbon and nitrogen sources, substrates and incubation times were evaluated. RESULTS: Maximum lipase activity was achieved after 24 hr of incubation with 1.5% of glucose syrup (1600±69.1 u/mg), 1% of fish powder (1238±36.7 u/mg) and olive oil (1407±2.1 u/mg) as low cost carbon and nitrogen sources and substrate, respectively. CONCLUSION: Our results show a significant increase in lipase activity with usage of low cost sources; this could help in reducing the media prices for industrial application of lipase enzyme.

Lack of association between rs1800795 (-174 G/C) polymorphism in the promoter region of interleukin-6 gene and susceptibility to type 2 diabetes in Isfahan population.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is an inflammatory autoimmune disease that mostly affects older adults. The etiology of T2DM includes both genetic and environmental factors. rs1800795 (-174 G/C) single nucleotide polymorphism (SNP) linked with autoimmune disorders predispositions, identified by Genome-Wide Association Study among genes, which immunologically related is considerably over signified. The goal of this study was to evaluate the association between rs1800795 (-174 G/C) polymorphisms in the promoter of interleukin-6 (IL-6) gene with susceptibility to T2DM in a subset of the Iranian population. MATERIALS AND METHODS: In this case-control study, 120 healthy subjects and 120 patients with T2DM were included. Genomic DNA obtained from whole blood samples and the polymerase chain reaction was used to amplify the fragment of interest contain rs1800795 SNP, restriction fragment length polymorphism method was applied for genotyping of the DNA samples with NlaIII as a restriction enzyme. SPSS for Windows software (version 18.0, SPSS, Chicago, IL, USA) was performed for statistical analysis. RESULTS: No significant differences were found between healthy controls and T2DM patients with respect to the frequency distribution of the cytokine gene polymorphism investigated. Odds ratio, adjusted for sex, age, and smoking status has displayed similar outcomes. CONCLUSION: These results indicated that the rs1800795 SNP is not a susceptibility gene variant for the development of T2DM in the Isfahan population. Further studies using new data on complex transcriptional interactions between IL-6 polymorphic sites are necessary to determine IL-6 haplotype influence on susceptibility to T2DM.