Today, many valuable proteins can be obtained in sufficient amounts using recombinant DNA techniques. However, frequently the expression of recombinant proteins results in the accumulation of the ...product in dense amorphous deposits inside the cells, called inclusion bodies. The challenge then is to transform these inactive and misfolded protein aggregates into soluble bioactive forms. Although a number of general guidelines have been proposed, the search for proper reconstitution conditions can be very laborious and time consuming. Here, we suggest a new versatile approach for solubilization and refolding of inclusion body proteins using a water–sodium bis-2-ethylhexyl sulfosuccinate–isooctane reverse micellar system. Instead of amorphous aggregates, a transparent solution is obtained, where refolded protein is entrapped inside the micelles. The entrapped enzyme has native-like secondary structure and catalytic activity. This approach has been implemented with
Fusarium galactose oxidase and
Stigmatella aurantiaca putative galactose oxidase.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A new antigalloping device for overhead lines with bundle conductors was introduced ten years ago, after laboratory tests and observations on actual 400 kV lines. A systematic full-scale test was ...performed in Kazakhstan in order to better evaluate torsional damper and detuner (TDD) efficiency. This report details the test station and the results obtained over a several month period of testing and measurement. The tests were carried out thanks to the mutual efforts of the ESSP and the Kazakh Power Research Institute (KazNIIE) at the field tests stand located in Chokpar.
We considered α-chymotrypsin (CT) in homogeneous water–organic media as a model system to examine the influence of enzyme chemical modification with hydrophilic and hydrophobic substances on its ...stability, activity and structure. Both types of modifying agents may lead to considerable stabilization of the enzyme in water–ethanol and water–DMF mixtures: (i) the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value is broadened and (ii) the range of organic cosolvent concentration at which the residual enzyme activity is observed is increased. We found that for both types of modification the stabilization effect can be correlated with the changes in protein surface hydrophobicity/hydrophilicity brought about by the modification. Circular dichroism studies indicated that the effects of these two types of modification on CT structure and its behavior in water–ethanol mixtures are different. Differential scanning calorimetry studies revealed that after modification two or three fractions or domains, differing in their stability, can be resolved. The least stable fractions (or domains) have properties similar to native CT.
Formation of enzyme–oligoamine complexes was suggested as an approach to obtain biocatalysts with enhanced resistance towards inactivation in water–organic media. Complex formation results in ...broadening (by 20–40% v/v ethanol) of the range of cosolvent concentrations where the enzyme retains its catalytic activity (stabilization effect). At moderate cosolvent concentrations (20–40% v/v) complex formation activates the enzyme (by 3–6 times). The magnitude of activation and stabilization effects increases with the number of possible electrostatic contacts between the protein surface and the molecules of oligoamines (OA). Circular dichroism spectra in the far-UV region show that complex formation stabilizes protein conformation and prevents aggregation in water–organic solvent mixtures. Two populations of the complexes with different thermodynamic stabilities were found in α-chymotrypsin (CT)–OA systems depending on the CT/OA ratio. The average dissociation constants and stoichiometries of both low- and high-affinity populations of the complexes were estimated. It appears that it is the low-affinity sites on the CT surface that are responsible for the activation effect.
A protein with a molecular weight of 70 kDa was isolated from bovine blood serum and purified to a homogenous state. This protein reversibly inhibited the adhesive serum glycoprotein with a molecular ...weight of 12 kDa, which displayed biological activity at ultralow doses. Amino acid analysis showed that the protein inactivator belongs to the group of prealbumins from vertebrate blood serum. The secondary structure of its molecule was characterized by a considerable number of α-helices. The conditions for inactivation of serum glycoprotein were studied. The interaction between the serum glycoprotein and the protein inactivator occurred over a long period of time (1 day). It should be emphasized that the presence of calcium ions was a necessary condition for the inactivation of the serum glycoprotein. The data suggest that inactivation of serum glycoprotein results from the formation of a molecular complex consisting of the protein inactivator and the glycoprotein, which is related to the carbon-protein interaction.PUBLICATION ABSTRACT
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The amino acid composition, structure, and physicochemical properties of a low-molecular-weight glycoprotein from cattle blood serum (SGP) were studied. The content of carbohydrates (represented by ...mannose-rich oligosaccharides) amounted to 45-50 wt %. The value of the specific partial heat of SGP, measured by differential scanning calorimetry (DSC), equaled 1.8 J/(g K), which is characteristic of unfolded proteins. Circular dichroic (CD) spectra of SGP led us to conclude that it is not highly structured and that it occurs in the shape of a statistical globule. The protein was deglycated using anhydrous trifluoromethane sulfonate (TFMS), after which its amino acid composition and the sequence of a fragment were determined. The results indicate that SGP is a protein not studied previously.PUBLICATION ABSTRACT
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Glycosylation procedures and their application for the elucidation of glycoprotein structure and structure-function relations, as well as for the development of analytical systems for clinical ...practice are reviewed. For some common cases found in research practice, the choice of optimal deglycosylation methods is discussed. Current views on the primary structure of glycoproteins are described briefly.
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