Ballast Proteins Research Articles

Оптимизация условий выделения IgY из желтка куриных яиц

Background . Immunoglobulins Y (IgY), obtained as a result of immunization of poultry against a specific pathogen, are highly active against this pathogen. Currently, passive immunization using IgY is very promising due to the lack of reactivity of IgY with respect to mammalian Fc receptors, low cost and ease of isolation. Egg yolk is a rich source of IgY, the total content of which exceeds 100 mg per chicken egg. Due to the significant differences between blood serum and egg yolk, the isolation of immunoglobulins from the latter requires specific purification from the lipid part of the yolk. This is achievable through a two-fold treatment, including the procedure for separating the water-soluble fraction (WF) from the lipid matrix at pH 4-5 and isolating IgY from the WF. Purpose . The purpose of this work is to optimize the conditions for the isolation of IgY, which would allow the implementation of a variant of deep processing of egg yolk. Materials and methods . To achieve this goal, chicken eggs were taken as an object of study, and statistical (RCCP), analytical (biuret), physicochemical (SDS-PAGE, ultrafiltration) methods were used. Results . The following conditions for IgY isolation were selected: freezing of the yolk solution in a mixture of sodium phosphate buffer: water acidified to pH 5.0 in a ratio of 1:6 at a temperature of -20°C and decanting from lipid components by filtration during spontaneous thawing at room temperature. The resulting water-soluble fraction was then subjected to precipitation with sodium chloride at a concentration of 10 wt. % and subsequent concentration on the UAM-10 membrane, which made it possible to achieve the content of the main substance (IgY) of at least 95% on a dry matter basis. Conclusions . The conditions for the selective isolation of IgY from egg yolk by optimizing the process were established: the dilution ratio of the yolk suspension is 6 and the concentration of the added NaCl salt to the water-soluble fraction is 10 wt. %; as a result, a regression equation Y=8.1834X_1+5.5258X_2+0.6005X_1^2+0.2819X_2^2 was obtained, which provides the maximum degree of purification of the target product from ballast proteins and impurities, which makes it possible to obtain an IgY-containing fraction with a protein content , varying in the range of 11.5 - 12.1 g / l and a purity of at least 95%.

Application of Ion Exchange Chromatography in the Development of Technology to Obtain Inactivated Poliovirus Vaccine

Relevance. The production and quality control of any drugs are strictly regulated. In the case of antiviral vaccines, the requirements for their safety and protective activity are getting tougher every year. Vaccine manufacturers face three tasks: ensuring high immunogenicity, safety and availability of the drug. During the development and production of immunobiological drugs, manufacturers must demonstrate sufficient purification from technological impurities to ensure the purity of the drug. Technological impurities directly depend on the technological process and the expression systems used. The Vero cell line has been widely used in the production of various antiviral vaccines for many decades. Thus, the improvement of technological processes for the purification of vaccine preparations from proteins and DNA of Vero cells is still matter of current interest.Aims. Selection of resins and reagents for ion exchange chromatography to reduce the amount of technological impurities in the inactivated polio vaccine production.Materials and Methods. To obtain viral suspensions, producer cultures were infected with poliovirus type 1 (Sabin strain LSc 2ab), type 2 (Sabin strain P712 Ch 2ab), and type 3 (Sabin strain Leon 12a1b). Multiplicity of infection was 0.02 ± 0.01 TCD50/cell. To evaluate the efficiency of ion-exchange chromatography we determined the degree of purification of fractions from ballast proteins, the degree of purification of fractions from residual cellular DNA, and the degree of extraction of the target antigen using specific formulas.Results and discussion. More than 80 experiments have been performed to purify type 1, type 2, and type 3 poliovirus concentrates using various sorbents. In quality control of purified concentrates of type 1, type 2 and type 3 polioviruses, in addition to analysis for total protein, an analysis was performed for the presence of Vero cell proteins.Conclusion. The use of the proposed modifications of purification of concentrates of Sabin strains of poliovirus types 1, 2 and 3 using ion exchange chromatography allows to obtain inactivated viral products that meet the requirements of WHO and the European Pharmacopoeia both in biochemical parameters (the content of host-cell DNA and the content of ballast proteins, including host-cell proteins) and specific activity (D-antigen content). Furthermore, additional purification of vaccines using ion exchange chromatography allows to reduce the content of residual cellular DNA to almost zero, which makes the inactivated polio vaccine the most attractive for its inclusion in various combined vaccines.

ISOLATION AND PURIFICATION OF PROTEIN THAT IS IMMUNOLOGICALLY SIMILAR TO HUMAN LACTOFERRIN

The study aimed at isolating a substance that is immunologically similar to human lactoferrin, hereinafter — microbial-derived lactoferrin (MdLF) isolated from K.pneumoniae liquid culture. Protein extraction started with isolation of ballast proteins in 2M ammonium sulphate (p.a.). Ion chromatography with cation-exchange agents was the basic method used for isolating MLF. Proteins from the isolated protein fractions were identified with AGID method. The isolated MLF is glucoprotein with the molecular mass of about 84,000, the agarose diffusion coefficient of 3.1 × 10–7 cm2 sec–1 and the relative electrophoretic mobility of 0.42. MLF is characterized by low hydrophobicity and elutes from phenylSepharose with 0.4 ammonium sulphate. Its isoelectric point equals to 9.2.

Indirect determination of FMDV 146S component concentration in non-inactivated suspension by comparison of graphs of the second derivative for real-time RT-PCR curves

During reproduction in biological systems, FMD virus forms four variants of components, three of which do not include RNA of the virus. In the process of industrial production of FMD vaccines, special attention is paid to the number of whole virions, which have the most important biological properties of FMD virus and are the main components that determine the immunogenicity of vaccine preparations. Raw materials for vaccines at various stages of the technological process are tested for concentration of FMDV 146S component. The traditional method of determination is quantitative complement fixation test. In recent years, real-time RT-PCR has been used for indirect determination of FMDV 146S component concentration in a virus-containing suspension. The article presents a new approach to indirect determination of FMDV 146S component concentration in a non-inactivated suspension by comparing the maximum extreme points of the graphs of the second derivative of the fluorescence signal accumulation curves and the number of amplification reaction cycles. The dependence between FMDV 146S component concentration and the maximum extreme points of the graphs of the second derivative of the fluorescence signal accumulation curve is presented in the form of a square function: C 146S FMDV = 0.0111( C p ) 2 – 1.0157 C p + 20.446 with a high accuracy of approximation (R 2 = 0.993). The proposed model allows to quantitatively estimate the content of 146S component in virus-containing vaccine raw materials. The presented method allows studying a large number of samples of non-inactivated raw materials for FMD vaccine in 4–5 hours. The main advantage of the proposed method is the capacity to determine the concentration of FMDV 146S component in a suspension with a high level of ballast proteins (more than 7.00 mg/cm 3 ) and complete viral particles (from 0.01 to 5.00 μg/cm 3 ).

Effectivity of tyrosinase purification by membrane techniques versus fractionation by salting out

The main goal of this study was to select micro- and ultrafiltration membranes that can be used for the purification of mushroom tyrosinase, replacing salting-out dual-step processes followed by centrifugations. In experiments, a raw extract from white mushrooms was used with high level of ballast proteins and brownish impurities. Four microfiltration membranes for the removal of undesired high molecular weight compounds were screened and that made of nitrocellulose was selected due to high recovery of enzymatic activity. Then diafiltration and concentration on the membrane made of polyethersulphone (300 kDa) was selected to recover 8% of proteins and 58% of tyrosinase activity with five- to seven purification fold, 10% of proteases, and 8% of brown impurities. It was shown that tyrosinase can be pre-purified by selected membranes yielding the enzyme quality at least comparable to that after double salting-out method but in one device. In both cases, subsequent purification by ion-exchange chromatography slightly increased purification degree of the enzyme and brown impurity removal. The surplus of membrane pre-purification is substantially higher thermal stability of the enzyme, enlarged after the chromatographic step, due to very low content of proteolytic enzymes.

Study of hydrophobin-type proteins from Funalia trogii

Hydrophobins are low-molecular surface-active proteins of fungi with high surface activity and the ability to self-assemble at the interface. The unusual properties of hydrophobins open up possibilities for their application in various fields, including medicine and the food industry. The wide range of possible applications of hydrophobins makes it important to develop and improve technology for their isolation and purification. The aim of the study was to select methods for the extraction of hydrophobin-type proteins and to study the ability of the obtained extracts to modify the solid surface. The source of hydrophobins in this study was the biomass of the fungus Funalia trogii. Methods for the isolation of hydrophobin-type proteins were developed, including purification of the extract from ballast proteins, followed by the destruction of agglomerates of hydrophobin-type proteins using acids in high concentrations. The surface activity and the ability to modify the surface of the obtained proteins were evaluated. As a result, we obtained extracts containing hydrophobin-type proteins with high surface activity. Funalia trogii extracts are capable of changing the hydrophobicity of the surface and can be used in various industries.

The Effect of Hypothermia on Some Structural and Functional Characteristics of Lactate Dehydrogenase of the Rat Brain

Abstract—We studied the activity and some physicochemical parameters of LDH in the rat brain under normal and hypothermic conditions. It was found that during hypothermia the activity of LDH in the rat brain increases, while this increase is less pronounced in an enzyme preparation purified from ballast proteins. A study of LDH self-fluorescence showed a decrease in the intensity of total fluorescence of LDH, in which the main contribution is made by tryptophan residues. Analysis of the second derivatives of the fluorescence spectra showed that hypothermia affects tryptophanyls, which are located on the periphery of the LDH molecule. Study of the binding kinetics of the fluorescent probe ANS with LDH showed the presence of at least two types of binding sites of the probe, which differ in polarity. Hypothermia leads to a decrease in the fluorescence intensity of the ANS, a decrease in the number of binding sites, an increase in the probe dissociation constants, and a shift in the inflection position on the graph of the temperature dependence of the ANS fluorescence to higher temperatures. The study of the content of sulfhydryl and carbonyl groups in the LDH molecule suggests that one of the causes of the changes in the activity and spectral characteristics of LDH in the brain of hypothermic rats is the modification of the enzyme by reactive oxygen species.

PRODUCTION OF PURIFIED FIBRINOGEN CONCENTRATE

Aim. To present a technique for obtaining fibrinogen concentrate purified from ballast proteins, which can be used in hemostatic drug production.Materials and methods. The proposed method is based on isolating fibrinogen from a cryoprecipitate solution by its precipitation with a polyethylene glycol solution followed by virus inactivation by a solvent/detergent. Purification from viral inactivation products and ballast proteins is performed by a two-step processing of the obtained fibrinogen concentrate with glycine solution.Results. The developed laboratory method for isolating fibrinogen has been optimized in terms of main parameters and scaled in pilot production.Conclusion. The presented laboratory technique for fibrinogen extraction is characterized by a high yield of the target product, thus being suitable for the production of hemostatic drugs.

Optimization of the refolding of recombinant human granulocyte-macrophage colony-stimulating factor immobilized on affinity sorbent

A method for the production and purification of biologically active recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), expressed in Escherichia coli, has been developed. Washing of an inclusion body fraction which allows the removal of numerous ballast proteins and on-column refolding of rhGM-CSF are specific characteristics of the method. The refolding efficiency reached 70%; the purity of the preparation constituted 95%. The biological activity was similar to those of other recombinant hGM-CSF analogs.

Magnetic porous corn starch for the affinity purification of cyclodextrin glucanotransferase produced by Bacillus circulans

Magnetic porous corn starch was prepared as an affinity adsorbent for the efficient and simple scale-up procedure for one-step purification of cyclodextrin glucanotransferase (CGTase) from Bacillus circulans. Magnetic affinity separation enabled isolation of CGTase from cultivation media (volumes between 10 and 400 mL) with ca 60–70% recovery after elution with alkaline buffers containing soluble starch; the enzyme purification factor was 19–25 in different batches. The majority of ballast proteins were removed during the purification process, which shows high selectivity of the affinity material used.

Use of cation exchange chromatography for human C-peptide isotope dilution – Mass spectrometric assay

An application of ion exchange chromatography for C-peptide analysis is described here. At the stage of C-peptide isolation, a strong cation exchanger (SP HP or MonoS) was used to purify the analyte from ballast proteins and peptides. The conditions of ion-exchange chromatographic separations were optimized using theoretical modeling of the net surface electric charge of the peptide as a function of pH. The purified and concentrated sample was further subjected to LC-MS/MS. In order to improve the reliability of analysis, two fragment ions were monitored simultaneously both for native C-peptide and internal standard, isotopically labeled C-peptides analogues (fragments with m/z of 927.7 and 147.2). Using ion-exchange chromatography, it became possible to process larger sample volumes, important for testing patients with very low C peptide levels, compared to currently used solid phase extraction methods.

Transition of recombinant allergens from bench to clinical application

The cloning and production of an increasing number of allergens through the use of DNA technology has provided the opportunity to use these proteins instead of natural allergen extracts for the diagnosis and therapy of IgE-mediated allergic disease. For diagnostic purposes, it is essential that the molecules exhibit IgE-reactivity comparable with that of the natural wild-type molecules, whereas T cell reactivity and immunogenic activity may be more important for allergen-specific immunotherapy. In relation to the latter, the development of hypoallergenic recombinant allergen variants is an approach which shows great promise. Clinical application of the proteins requires that they must be produced under conditions of Good Manufacturing Practice and meet the specifications set down in the appropriate Regulatory Guidelines, principally the ICH-Guidelines. Special consideration has to be given to the choice of expression system, the design of the expression vectors, and the purification strategy to obtain a pure product free from toxins and contamination. The availability of the pure recombinant molecules provides the opportunity to formulate preparations that are free from the non-allergenic ballast proteins present in natural allergen extracts and which contain relative concentrations of the allergens in clinically appropriate proportions.

Enzyme self-inactivation is a main limitation of the preparation of eicosanoids. Enzymatic synthesis of thromboxane and 12(S)-hydroxytetraenoic acid.

Synthesis of prostanoids is accompanied by various processes reducing the product yield. These processes are also known to affect syntheses of thromboxane (TX) and 12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12-HETE). Partially purified preparations of TX synthase and prostaglandin (PG) synthase were used to optimize TX synthesis with respect to concentrations of the enzymes and eicosapolyenoic acid (EPA). Conditions for the maximum product yield and the minimum consumption of enzymes were determined. Consumption of the TX synthase was large owing to its inactivation during the reaction and the nonenzymatic destruction of the intermediate product PG-endoperoxide. Separate addition of PG and TX synthases increased the product yield by preventing EPA sorption on ballast proteins. Microsomal 12-lipoxygenase (12-LO) was also shown to be inactivated during the reaction, and this process was the major limitation of 12-HETE synthesis. Lipoxygenase reaction in the presence of some reducing agents led to a considerable increase of the 12-HETE yield, supposedly by preventing further oxidation of the 12-LO reaction product 12-hydroperoxy derivative of eicosatetraenoic acid. The possibility of using human blood platelet microsomes for preparation of some derivatives of EPAs is discussed.

Rapid isolation of bacterial proteinases by column chromatography on sawdust

AbstractProteolytic enzymes produced by some strains of Bacillus sp. were isolated by column chromatography on sawdust. Ballast proteins were eluted with water while the adsorbed proteinases were eluted with 1 m sodium chloride solution. The specific activities of proteinases increased 54‐ to 70‐fold after chromatography.

Isolation and study of the protease-inhibitor system of wheat grain

A modified method for obtaining a protease and its natural protein inhibitor from the endosperm of wheat grain is proposed. The enzyme is purified by additional precipitation of part of the ballast proteins with ammonium sulfate, and the inhibitor by two successive precipitations of the bulk of the inactive protein with the aid of TCA of different concentrations. The specific activity of the protease increases by a factor of 5–6, and the activity of the inhibitor by a factor of 3–4. Two active isoenzymes of protease have been found in each of the varieties of wheat investigated by disk eletrophoresis.

Recovery of Blood Group Antibodies from Erythrocyte Powder

Abstract A powder from dried human erythrocyte stromata was prepared to determine whether normal autoantibodies could be recovered from unsensitized erythrocyte powder and to study some of the properties of anti-A, anti-B and anti-Rho(D) antibodies recovered from sensitized powder. Twenty-five samples of unsensitized erythrocyte powder of all ABO blood groups were tested. The amount of eluted powder was as great as 200 mg., representing about 25 ml. of packed red cells. In no instance could antibody be detected in the eluates. This negative finding could be explained in three ways: by lack of antibody production, in disagreement with Landsteiner’s rule; by neutralization of antibody by a corresponding antigen before it reached the circulation; or by immediate elimination of sensitized erythrocytes from the circulation. The eluate of A erythrocyte powder sensitized with O serum agglutinated both A and B red cells. After neutralization with A polysaccharides, the anti-A antibody disappeared and anti-B remained. These experiments appear to support the theory of the multispecific character of natural antibodies. Rh-positive erythrocyte powder was sensitized with an incomplete anti-D (anti-Rho) serum and eluted in saline. The eluate did not agglutinate Rhpositive cells, but sensitized them for the action of Coombs’ serum. The mixture of Coombs’ serum with the eluate also produced agglutination of Rh-positive cells. These phenomena are explained as being due to the lack of or the very small amount of ballast proteins in the saline eluate.