The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology

The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.     

Acute creatine administration improves mitochondrial membrane potential and protects against pentylenetetrazol-induced seizures

A growing body of evidence indicates that creatine (Cr) exerts beneficial effects on a variety of pathologies where energy metabolism and oxidative stress play an etiological role. However, the benefits of Cr treatment for epileptics are still shrouded in controversy. In the present study, we found that acute Cr treatment (300 mg/kg, p.o.) prevented the increase in electroencephalographic wave amplitude typically elicited by PTZ (30, 45 or 60 mg/kg, i.p.). Cr treatment also increased the latency periods of first myoclonic jerks, lengthened the latency periods of the generalized tonic–clonic seizures and reduced the time spent in the generalized tonic–clonic seizures induced by PTZ (60 mg/kg). Administration of PTZ (all doses) decreased Na⁺, K⁺-ATPase activity as well as adenosine triphosphate (ATP) and adenosine diphosphate levels in the cerebral cortex, but Cr treatment prevented these effects. Cr administration also prevented increases in xanthine oxidase activity, adenosine monophosphate levels, adenosine levels, inosine levels and uric acid levels that normally occur after PTZ treatment (60 mg/kg, i.p.). We also showed that Cr treatment increased the total Cr (Cr + PCr) content, creatine kinase activity and the mitochondrial membrane potential (ΔΨ) in the cerebral cortex. In addition, Cr prevented PTZ-induced mitochondrial dysfunction characterized by decreasing ΔΨ, increasing thiobarbituric acid-reactive substance levels and increasing protein carbonylation. These experimental findings reinforce the idea that mitochondrial dysfunction plays a critical role in models of epileptic seizures and suggest that buffering brain energy levels through Cr treatment may be a promising therapeutic approach for the treatment of this neurological disease.     

Simultaneous Saccharification and Fermentation of Hydrothermal Pretreated Lignocellulosic Biomass: Evaluation of Process Performance Under Multiple Stress Conditions

Industrial lignocellulosic bioethanol processes are exposed to different environmental stresses (such as inhibitor compounds, high temperature, and high solid loadings). In this study, a systematic approach was followed where the liquid and solid fractions were mixed to evaluate the influence of varied solid loadings, and different percentages of liquor were used as liquid fraction to determine inhibitor effect. Ethanol production by simultaneous saccharification and fermentation (SSF) of hydrothermally pretreated Eucalyptus globulus wood (EGW) was studied under combined diverse stress operating conditions (30–38 °C, 60–80 g of liquor from hydrothermal treatment or autohydrolysis (containing inhibitor compounds)/100 g of liquid and liquid to solid ratio between 4 and 6.4 g liquid in SSF/g unwashed pretreated EGW) using an industrial Saccharomyces cerevisiae strain supplemented with low-cost byproducts derived from agro-food industry. Evaluation of these variables revealed that the combination of temperature and higher solid loadings was the most significant variable affecting final ethanol concentration and cellulose to ethanol conversion, whereas solid and autohydrolysis liquor loadings had the most significant impact on ethanol productivity. After optimization, an ethanol concentration of 54 g/L (corresponding to 85 % of conversion and 0.51 g/Lh of productivity at 96 h) was obtained at 37 °C using 60 % of autohydrolysis liquor and 16 % solid loading (liquid to solid ratio of 6.4 g/g). The selection of a suitable strain along with nutritional supplementation enabled to produce noticeable ethanol titers in quite restrictive SSF operating conditions, which can reduce operating cost and boost the economic feasibility of lignocellulose-to-ethanol processes.     

Polystoma knoffi n. sp. and Polystoma travassosi n. sp. (Monogenea: Polystomatidae): naming museum-archived specimens from Brazil

Systematic Parasitology - In 1978, Kohn and co-workers deposited several polystome (Monogenea) specimens infecting several Brazilian anurans [Trachycephalus mesophaeus (Hensel), T. nigromaculatus...     

Protein extraction and comparison of stain protocols for analysis of two-dimensional electrophoresis gels

Protein extraction and the proteome of Lactobacillus delbrueckii subsp. bulgaricus were studied using different stains. The reversible silver staining technique was shown to be more sensitive than the irreversible silver stain. Coomassie colloidal was demonstrated to be as sensitive as reversible silver stain; however, the Coomassie colloidal blue solution developed a higher background and for sample preparation was more time-consuming.     

Structural localization of disease-associated sequence variations in the NACHT and LRR domains of PYPAF1 and NOD2

Several autoinflammatory diseases with distinct clinical manifestations have been associated with sequence variations in the gene products PYPAF1/CIAS1 and NOD2/CARD15. Both proteins belong to the PYD/CARD-containing family of apoptosis regulators and activators of pro-inflammatory caspases. To gain insight into the dysfunctional role of sequence alterations, we assembled a structure-based multiple sequence alignment of family members and related proteins. This allowed us to analyze the putative effect of the alterations on the function of nucleotide-binding (NACHT) and leucine-rich repeat (LRR) domains shared by the family members. In support of this analysis, we carefully selected template structures for the NACHT and LRR domains and mapped the genetic variations onto 3D domain models. Additionally, we propose a model of the NACHT and LRR domain complex. Our study revealed that many of the disease-associated sequence variants are located close to highly conserved sequence regions of functional relevance and are spatially adjacent in the predicted 3D structure. The implications on the domain functions such as NTP-hydrolysis or oligomerization are discussed.