Production of Recombinant Polypeptides Binding α2-Macroglobulin and Analysis of Their Ability to Bind Human Serum α2-Macroglobulin.

Many streptococcal strains bind to two main human blood plasma proteins: IgG and human serum albumin (HSA). Protein G expressed in group C and G streptococci has specific binding regions for these proteins. Protein G in group G streptococcal strains also contains a region binding another human plasma protein, α2-macroglobulin (α2-М), upstream to the HSA-binding domain. Two recombinant polypeptides GM and GM1 capable of binding to α2-М were obtained using the G4223 strain of a group G Streptococcus, protein G molecule of which interacts with three human blood serum proteins (IgG, HSA, and α2-М). However, polypeptide GM containing three IgG-binding and three HSA-bindings domains and the region binding α2-М has higher molecular mass and higher affinity to α2-М than polypeptide GM1 that includes only the α2-М binding region.

Expression of New Recombinant IgG-Binding Polypeptides and Analysis of Their Capacity to Bind Human IgG.

Protein G is present in group G streptococcus strain (G4223); the IgG-binding part of this protein contains three IgG-binding domains and binds human IgG with very high activity. We obtained two recombinant polypeptides G4223 and G14223 with high IgG-binding activity. Polypeptide G14223 consisting of three IgG-binding domains and W region has higher molecular weight and is characterized by higher affinity for IgG than polypeptide G4223 consisting of only three IgG-binding domains. It was shown that polypeptide affinity depends on its structure and size.

Production of Recombinant Polypeptides Containing One GA-Module and Analysis of Their Ability to Bind to Human Albumin.

Surface proteins of many bacterial species interact with human serum albumin (HSA) via a special region of amino acid sequence termed GA module. For instance, surface peptostreptococcal albumin-binding protein of anaerobic bacteria Peptostreptococcus magnus contains one HSA-binding GA-module. Protein G from group G and C Streptococcus strains isolated from humans has HSA-binding region consisting of three GA-modules. HSA-binding protein containing two GA-modules was found in strains of group G Streptococcus of animal origin. We obtained two recombinant polypeptides GA1 and GA2 congaing one GA-module each. Recombinant polypeptide with two GA-modules binds HSA with a much higher affinity than polypeptides GA1 and GA2 containing one GA-module. Polypeptide with the second GAmodule more effectively binds HSA than polypeptides with the GA-module.

[The test-system for detection of microalbuminuria using the new recombinant albumin-bounded poly-peptide].

The diabetes mellitus and arterial hypertension are among the most significant pathologies conditioning disorder of excretion of protein with urine. These very diseases are mostly dangerous for kidneys. Therefore, important significance has the search of early manifestations of damage of kidneys in patients with these diseases. The microalbuminuria is one of early manifestations of affection of kidneys in patients with diabetes mellitus and arterial hypertension. Only this early (pre-clinical) stage of affection of kidneys is the only reversible one in case of prescription of medicinal therapy. Nowadays, factually all applied diagnostic test-systems for detection ofmicroalbuminuria are based on immunological half-quantitative and quantitative detection of concentration of human serum albumin in urine. In this study was applied new recombinant human serum poly-peptide A3 from strain of streptococcus group G isolated from cow milk. The human serum albumin-binding capacity of poly-peptide A3 was analyzed in comparison with poly-peptide A2. Previously, recombinant human serum albumin-binding poly-peptide A2 was primarily applied in test-system for detection of microalbuminuria instead of commonly used antibodies. The analysis of 'human serum albumin-binding capacity of recombinant human serum poly-peptide A3 and A2 demonstrated that both of them can interact with human serum albumin in solution and adsorbed condition. This characteristic permitted applying poly-peptide A3 in immobilized form in qualitative test-system for detecting microalbuminuria. The actual study also propose specific and sensitive technique of screening and monitoring of patients with diabetes mellitus and arterial hypertension. The mentioned technique used tagged human serum albumin-binding polypeptide A3 combined with microchip technology. The comparison of test-systems using recombinant poly-peptides A3 and A2 established that application of poly-peptide A3 in test-system permits to detect more precisely concentration of human serum albumin in urine samples. The test-system of this kind was successfully implemented for both detection and qualitative identification of microalbuminuria in patients with diabetes mellitus and arterial hypertension.

The Relationship between Albumin-Binding Capacity of Recombinant Polypeptide and Changes in the Structure of Albumin-Binding Domain.

Many bacteria express surface proteins interacting with human serum albumin (HSA). One of these proteins, PAB from anaerobic bacteria, contains an albumin-binding domain consisting of 45 amino acid residues known as GA domain. GA domains are also found in G proteins isolated from human streptococcal strains (groups C and G) and of albumin-binding protein isolated from group G streptococcal strains of animal origin. The GA domain is a left-handed three-helix bundle structure in which amino acid residues of the second and third helixes are involved in albumin binding. We studied the relationship between HSA-binding activity of the recombinant polypeptide isolated from group G streptococcus of animal origin and structure of the GA domain is studied. Structural changes in GA domain significantly attenuated HAS-binding capacity of the recombinant polypeptide. Hence, affinity HSA-binding polypeptide depends on stability of GA domain structure.

[A New Approach to the Depletion of Albumin and Immunoglobulin G from Human Serum].

The use of proteomic analysis to find potential diagnostic biomarkers is limited by the presence of serum albumin (HSA) and immunoglobulin (IgG) at high concentrations in patients' blood; these substances impede the detection of serum proteins with similar molecular weights. Recombinant HSA- and IgG-binding polypeptides are used as ligands in creating sorbents for complete removal of the proteins by affinity chromatography. The binding specificity of the sorbents for HAS and IgG is higher than that of the conventionally used antibodies. A composite sorbent enabling the depletion of HSA and IgG from serum by single-step affinity chromatography is obtained. The. developed sorbents were used to prepare serum for proteomic analysis.

[Intranasal co-administration of recombinant streptococcus group B polypeptides and influenza deltaNS1 vaccine].

In the present work, the immunoadjuvant properties of the influenza deltaNS1 vaccine virus after intranasal administration in combination with recombinant GBS polypeptides was tested in mice. According to our data, co-administration of recombinant GBS polypeptides and influenza deltaNS1 vaccine resulted in the increase in the immunogenicity and protective efficacy of bacterial proteins. Combined vaccination with the GBS polypeptides and influenza deltaNS1 vaccine has a potential to be used not only for prophylaxis infections caused by SGB, but also for prevention of the bacterial complications of influenza.

[Immunogenicity of modified by dextran recombinant fragment of Bac protein of group B streptococci].

Opportunity to increase of immunogenicity of recombinant polypeptide P6 constructed on the basis of surface protective Bac protein by its chemical conjugation with dextran (D) 40 was studied. 3 preparations with different quantity of protein and polysaccharide components were obtained. Their testing with standard serum showed that antigenic determinants of the polypeptide were preserved although partly enclosed and structure of antigenic determinants did not significantly changed. On the model of subcutaneous immunization of mice it has been shown that two preparations--P6D2 and P6D3--have improved immunological characteristics. Conjugation of polypeptide P6 with dextran let to increase of immune response to P6 and affinity of P6-specific antibodies. Injection of nonconjugated P6 and dextran mixture showed that free dextran is not immunogenic and it suppress synthesis of P6-specific antibodies without effect on their affinity. Intranasal administration of nonconjugated P6 did not lead to P6-specific IgG in serum. After conjugation with dextran polypeptide P6 was recognized as an antigen and stimulated production of small quantity of antibodies. Technological process of chemical binding of protein antigen with polysaccharides, which let to regulate protein and polysaccharide components ratio, can be the effective method to increase immunogenicity of recombinant polypeptides.

[The light diffusion technique for estimation of the size of urine particles in patients with type 2 diabetes mellitus in the Tamm-Horsfall protein precipitation].

Tamm-Horsfall uroprotein accounts for more than 50% of the urinary proteins in healthy individuals. In abnormalities, it creates a favorable background for detecting smaller-sized uroproteins and for diagnosing pathological processes from the results of native urine tests. In this connection, there is a need for precipitating Tamm-Horsfall glycoprotein while applying laser correlation spectroscopy to analyze the size of urine particles in patients with type 2 diabetes mellitus. Eighty patients with this condition concurrent with different stages of diabetic nephropathy and 23 apparently healthy individuals were examined. The findings suggest that the subfraction urine composition before and after Tamm-Horsfall protein precipitation is different in apparently healthy individuals and patients with type 2 diabetes mellitus concurrent with diabetic nephropathy. This is most likely to be due to the change in the qualitative composition of protein as renal lesion progresses, to the specific features of protein excretion at different stages of a pathological process, and to different concentrations of other low and high molecular-weight proteins.

Quantitative investigation of the affinity properties of different recombinant forms of protein G by means of high-performance monolithic chromatography.

The recombinantly produced different forms of protein G, namely monofunctional immunoglobulin G (IgG) binding, monofunctional serum albumin (SA) binding and bifunctional IgG/SA binding proteins G, are compared with respect to their specific affinities to blood IgG and SA. The affinity mode of the recently developed high-performance monolithic disk chromatography has been used for fast quantitative investigations. Using single affinity disks as well as two discs stacked into one separation unit, one order of magnitude in adsorption capacities for IgG and SA were found both for monofunctional and bifunctional protein G forms used as specific affinity ligands. However, despite the adsorption difference observed, the measured dissociation constants of the affinity complexes seemed to be very close. The analytical procedure developed can be realized within a couple of minutes. Up-scaling of the developed technology was carried out using another type of monolithic materials, i.e. CIM affinity tubes.

[Comparative characteristics of protein G gene in Streptococci of groups C and G isolated from humans]. / Sravnitel'naia kharakteristika izoliatov streptokokkov grupp C i G, vydelennykh ot cheloveka, po genu G belka.

The treatment of streptococci, groups C and G, with bromocyanogen made it possible to isolate surface G protein, capable of binding human serum albumin (HSA) and polyclonal IgG. In this work the presence of G protein in all staphylococcal strains, groups C and G, is shown. The differences between the strains by the level of expression, molecular weight and functional activity of G protein, extracted from streptococci of groups C and G, permitted the identification of 3 groups of strains, containing the molecules of G protein with different numbers of IgG- and HSA-binding domains: with 3 IgG- and HSA-binding domains, with 2 IgG- and HSA-binding domains and with only 2 IgG-binding domains. Each strain under study expressed only one of the molecule of G protein. The work shows the possibility of the identification of streptococci, groups C and G, by the molecular characteristics of G proteins themselves and their respective coding genes.

[The detection of microalbuminuria by using a recombinant albumin receptor]. / Opredelenie mikroal'buminurii s primeneniem rekombinantnogo al'buminovogo retseptora.

Methods of ELISA competitive binding and blotting on nitrocellulose membranes were developed for detecting microalbuminuria in diabetic nephropathy. These methods are based on the use of recombinant albumin receptor. They are highly specific and sensitive and are recommended for everyday clinical use.

[Obtaining and properties of recombinant protein G]. / Poluchenie i svoistva rekombinantnogo belka G.

A gene for G protein from Streptococcus strain G148 was cloned in Escherichia coli, which gave rise to several plasmids. One plasmid containing a 1.5 kb insert coding for entire G protein with 63 kD. This protein had both an IgG binding capacity and albumin-binding activity. The second plasmid containing a 0.7 kD insert coded for protein with MM of 38 kD and had only an IgG-binding activity. The third coding for protein with 25 kD has only albumin-binding activity. After subcloning the 1.5-kb insert into the other vector pSP65 and analysing the nucleotide sequence of this insert both in pSP65 vector, the authors came to the conclusion that the proteins obtained are fusion protein of G protein and beta-galactosidase. All the proteins were prepared by affinity chromatography on IgG sepharose or on HSA sepharose. The interaction between G protein and polyclonal and monoclonal IgG of the reactions between G protein and human IgG have determined.

[Creation and analysis of a bank of chromosomal genes of Streptococcus group A]. / Sozdanie i analiz banka khromosomnykh genov Streptokokka gruppy A.

The representative genomic library of chromosomal genes has been constructed for streptococcus group A serotype M48 strain 1/64 on the vector lambda L 47.1. Screening of the obtained genomic library by hybridization and immunological techniques revealed about 50 clones producing the streptococcal antigens (extracellular nonidentified products and non-type specific structural streptococcal proteins). Among the recombinant clones three were found to harbour the genetic determinants for M-protein. One the clones contains a determinant coding for epitopes crossreacting with antisera to M-proteins of other serotypes and a protective epitope. The presence of the latter was tested in an indirect bactericidal test.

Características del bacteriófago virulento C1 de estreptococos del grupo C: II propiedades fisicoquimicas y moleculares del ADN del fago C1 / Characteristics of virulent C1 streptococci bacteriophague of group C: II physicochemical and molecular properties of the DNA of phague C1

El bacteriófago virulento C1 es capaz de reproducirse en estreptococos de los grupos C y A, bacterias capaces de causar enfermedades en animales y en el hombre. Un conocimiento mayor del fago C1 podría contribuir al estudio de los estreptococos correspondientes mediante el intercambio genético. Los resultados de este trabajo muestran que el ADN-C1 presenta una densidad de flotación de 1,705 g/cm a la tres-CsC1, temperaturas de fusión de 75 y 88-C. Siete de las 15 restrictasas incubadas con el ADN-C1 no exhibieron sitio de reconocimiento en su secuencia, el mapa físico fue construido para las ocho endonucleasas restantes. Fueron clonados los fragmentos B y B-C Hind III en pBR 322. Los estudios preliminares de estos clones no mostraron la presencia de LAF en los extractos bacterianos

Características del bacteriófago virulento C1 de estreptococos del grupo C. I: morfología y propiedades biomoleculares del fago C1 / Characteristics of the virulent bacteriophage C1 of group C streptococcus. I: morphology and biomolecular properties of C1 phage

El bacteriófago C1 específico para la cepa C4540 se ha utilizado para la producción de una lisozima asociada al fago (LAF), que tiene la propiedad de lisar estreptococos y otras bacterias. Sin embargo, poco se conoce de las características biomoleculares de este fago, que es capaz de reproducirse en otras cepas del grupo C de estreptococos, y del grupo A, bacterias capaces de producir enfermedades en animales y en el hombre. Mediante el intercambio genético entre el fago y los correspondientes estreptococos, podrían ser estudiados los factores genéticos de patogenicidad de estas bacterias, para lo cual se hace necesario el conocimiento de las características del propio bacteriofago. En el presente trabajo se estudiaron las características morfológicas, fisicoquímicas y biomoleculares de la partícula del fago C1 y sus proteínas. La microscopia electrónica muestra que el fago C1 posee una cabeza hexagonal de la cual surge un pequeño vástago constituido por 6-8 filamentos; la densidad de flotación de la partícula es de 1,462 g/cm a la 3 en CsC1; la cápsula del fago presenta siete polipéptidos electroforéticamente diferentes. Los datos evidenciaron la ausencia, en la estructura de la cápsula, de la potente lisozima asociada al fago C1, lo que contradice los dato de la literatura

Características de los bacteriófagos virulentos CAI y A25 de estreptococos del grupo A / Characteristic of CAI and A25 bacteriophages from group A streptococci

Los estreptococos del grupo A son capaces de provocar enfermedades en el hombre. El estudio genético de los factores de patogenicidad de estas bacterias ha estado limitado a la transducción (por bacteriófagos), sin embargo, las complejas características de los correspondientes bacteriófagos no se han estudiado suficientemente. En el presente trabajo se investigaron las características fisicoquímicas de las partículas, proteínas y ADN de los bacteriófagos CA1 y A25 de estreptococos del grupo A. Los principales resultados mostraron que el fago CA1 es idéntico morfológicamente al A25, presentando una cabeza hexagonal (60 nm) con una larga cola no contráctil (180 nm), según los datos de la microscopía electrónica. La estructura de la partícula de ambos fagos está constituida por 10 polipéptidos electroforéticamente diferentes. Los ADNs de ambos fagos presentan características similares: densidad de flotación 1,709 g/cm a la tres CsCI; dos zonas de temperatura de fusión a 80- y 88-C; ambos fagos presentan moléculas de ADN lineales y de doble hebra de una longitud de 38,3ñ 1,1 kb. Los datos de los análisis de homología mostraron una complementariedad total entre los ADNs de estos fagos. La secuencia de los ADNs de los bateriófagos A25 y CA1 contiene sitios de reconocimiento para seis endonucleasas (Hae II, Msp I, Ben I, Hind III y Bsu RI); basado en los análisis de restricción se construyó el mapa físico preliminar para ambos ADNs