Technical refolding of proteins: Do we have freedom to operate?

Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale production of therapeutic proteins that do not require post-translational modifications. High expression yields and simple recovery steps of inclusion bodies from the host cells are attractive features industrially. However, the value of an inclusion body-based process is dominated by the solubilization and refolding technologies. Scale-invariant technologies that are economical and applicable for a wide range of proteins are requested by industry. The main challenge is to convert the denatured protein into its native conformation at high yields. Refolding competes with misfolding and aggregation. Thus, the yield of native monomer depends strongly on the initial protein concentrations in the refolding solution. Reasonable yields are attained at low concentrations (≤0.1 mg/mL). However, large buffer tanks and time-consuming concentration steps are required. We attempt to answer the question of the extent to which refolding of proteins is protected by patents. Low-molecular mass additives have been developed to improve refolding yields through the stabilization of the protein in solution and shielding hydrophobic patches. Progress has been made in the field of high-pressure renaturation and on-column refolding. Mixing times of the denatured protein in the refolding buffer have been reduced using newly developed devices and the introduction of specific mixers. Concepts of continuous refolding have been introduced to reduce tank sizes and increase yields. Some of the patents covering refolding of proteins will soon expire or have already expired. This gives more freedom to operate.     

Use of the water-level fluctuation analysis tool (Regcel) in hydrological status assessment of Finnish lakes

Regulation of water flow constitutes the most important hydromorphological burden to Finnish lakes. The total area of regulated lakes is nearly 11,000 km², equalling one-third of the total area of Finnish inland waters. Extensive research projects have been carried out since the end of the 1980s to find out opportunities to mitigate harmful effects of the regulation of watercourses. A water-level fluctuation analysis tool, known as Regcel, has been developed to study water level data and to identify the most significant impacts. Results of the Regcel analysis give an overall picture of the impact of lake regulation in northern climate. The model is based on relationships between the water-level fluctuation and factors related to environmental, social and economical effects. Regcel has been used in 12 Lake Regulation Development Projects in Finland. In this article, we show how the Regcel model was applied in two cases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editors: K. M. Wantzen, K.-O. Rothhaupt, M. M?rtl, M. Cantonati, L. G.-Tóth & P. Fischer Ecological Effects of Water-Level Fluctuations in Lakes     

Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner

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The intranuclear bodies of Entamoeba

Intranuclear bodies which are spherical in shape are clearly seen by ‘Anoptral’ (negative) phase microscopy in the nucleus of Entamoeba. These bodies vary in size and numbers from cell to cell. With interference microscopy the intranuclear bodies appear as spherical granules in the nucleus of the cell. Their distribution and numbers are again very variable. With electron microscopy the bodies can be clearly seen inside the nucleus. They are always spherical in shape but vary in size from 0·1 to 1·5 μm. They may be empty or contain granular or membranous material. They display the capacity to move out of the nucleus.     

Neuro-epithelial bodies in organ cultures of fetal rabbit lungs

Lung explants from fetal rabbit at the late glandular stage of development (20 days' gestation) and near term (31 days' gestation) were maintained in organ culture for up to 22 days. They were studied by light and electron microscopy to determine whether neuro-epithelial bodies (NEB) of the lung retain structural integrity in vitro. Cultured NEB retained argyrophilia and specific amine fluorescence after formaldehyde condensation. Their ultrastructural morphology showed some differences from that of uncultured NEB: the terminal axons had degenerated and the secretory granules (dense-core vesicles, DCV) were slightly larger, more pleomorphic, more electron-dense, and redistributed throughout the cytoplasm rather than being confined chiefly to the basal regions. These changes, together with hypertrophy of Golgi zones, suggest increased synthesis and storage of secretory products in the DCV during culture. In NEB from near-term explants cultured for 7 days and incubated with reserpine, the core of DCV decreased in size and electron-density and became finely granular, a sign of amine release. Ca++ ionophore No. A-23187, also, induced changes in the ultrastructure of DCV, suggesting that the secretory process in lung neuro-endocrine cells, as in other secretory cells, is Ca++-dependent.     

Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development

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The SGLT2 inhibitor dapagliflozin promotes systemic FFA mobilization,enhances hepatic β-oxidation,and induces ketosis

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin (1 mg/kg/day, formulated in a water, PEG400, ethanol, propylene glycol solution, 4 weeks) on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10-³H(N)]-palmitic acid tracer by estimating rates of plasma FFA appearance (R_a), whole-body FFA oxidation (R_ox), and nonoxidative disposal (R_st). In the liver, clearance (K_β-ox) and flux (R_β-ox) of FFA into β-oxidation were estimated using [9,10-³H]-(R)-bromopalmitate/[U-¹⁴C]palmitate tracers. As expected, dapagliflozin induced glycosuria and a robust antidiabetic effect; treatment reduced fasting plasma glucose and insulin, lowered glycated hemoglobin, and increased pancreatic insulin content compared with vehicle controls. Dapagliflozin also increased plasma FFA, R_a, R_ox, and R_st with enhanced channeling toward oxidation versus storage. In the liver, there was also enhanced channeling of FFA to β-oxidation, with increased K_β-ox, R_β-ox and tissue acetyl-CoA, compared with controls. Finally, dapagliflozin increased hepatic HMG-CoA and plasma β-hydroxybutyrate, consistent with a specific enhancement of ketogenesis. Since ketogenesis has not been directly measured, we cannot exclude an additional contribution of impaired ketone body clearance to the ketosis. In conclusion, this study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward β-oxidation.     

High-throughput system for determining dissolution kinetics of inclusion bodies

Efficient solubilization is a crucial step during inclusion body processing and dissolving conditions were usually empirically established. Here we describe a new methodology for rapid screening of solubilization conditions and evaluation of dissolution kinetics in microtiter plates. Increase of protein in solution over time was directly related to decrease of turbidity measured by absorbance at 600 nm. Dissolution kinetics of inclusion bodies were described by a first-order reaction kinetics, which was used for drug dissolution modeling. Reaction constants were in the range of 0.01–0.03 s^–1 for buffer conditions providing sufficient solubilization power. This method is not limited to the screening of optimal buffer conditions for solubilization and can be applied for studying other parameters involved in the solubility of IBs, such as pI of the protein, influence of fermentation conditions, influence of initial protein concentration, and more.