Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models.

Recombinant bone morphogenetic protein-2 (rhBMP-2) has pronounced osteoinductive properties, as evidenced by the results of experimental and clinical practices. This applies to both the protein produced in eukaryotic cells and the protein synthesized in bacterial cells. In eukaryotic expression systems, production of the protein is extremely low and, consequently, the cost of materials on its basis is very high. Therefore, optimization of heterologous expression systems for rhBMP-2 production represents an important task. In the present work, optimization of codon composition of the rhBMP-2 gene nucleotide sequence and secondary structure of the transcript, as well as strain selection for efficient gene expression, were carried out. The producing strain based on Escherichia coli BL-21(DE3) provides a high level of rhBMP-2 synthesis (about 57% of total cell proteins). Biological activity of rhBMP-2 dimeric forms purified from the obtained producing strain was measured by induction of alkaline phosphatase activity in C2C12 cells. It is comparable with that of commercial rhBMP-2 expressed in E. coli (R&D Systems, United States). Purified rhBMP-2 does not contain impurities of E. coli endotoxin and can be used in experimental studies of osteoinduction in laboratory animals.

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second encoding a 749 aa multimodular endo-1,4-β-glucanase CelD (85019 Da). The N-terminal region of the protein includes a signal peptide and a catalytic module of glycoside hydrolase family 5 (GH5), followed by a carbohydrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic module and CBM28, were produced in E. coli cells and purified to homogeneity. An analysis of the catalytic properties showed CelD to be an endo-1,4-β-glucanase with maximum activity on barley β-glucan at pH 6.2 and 70°C. The enzyme was stable at 50°C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 was decreased on cellulose substrates, and its thermostability has dropped. Binding of CBM28 to amorphous cellulose has been almost irreversible as it could not be removed from this substrate in a range of pH of 4–11, temperatures of 0–75°C, and NaCl concentrations of 0–5 M. Only 100% formamide or 1% SDS have been able to remove the protein.

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second one of 749 aa encoding a multimodular endo-1,4-beta-glucanase CelD (85019 Da). N-terminal region of the protein includes the signal peptide and the catalytic module of glycoside hydrolase family 5 (GH5), followed by the substrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic one and CBM28, were produced in E. coli cells and purified to homogeneity. Analysis of the catalytic properties showed CelD to be endo-1,4-beta-glucanase whose maximum activity was exhibited on beta-glucan of barley at pH 6.2 and 70 degrees C. The enzyme was stable at 50 degrees C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 decreased on cellulose substrates, and its thermostability was dropped. Binding of CBM28 to amorphous cellulose was almost irreversible as it could not be removed from this substrate in a range of pH 4-11, temperatures--of 0-75 degrees C, and NaCl concentration--of 0-5 M. Only 100% formamide or 1% SDS were able to remove the protein.

The genes of ESAT-6, CFP-10 and Ag85A secreted antigens of Mycobacterium tuberculosis were cloned in Escherichia coli. Fusion proteins Esat6-CBD, Ag85A-CBD and CFP10-CBD with thermostable cellulose binding domain (CBD) of Anaerocellum thermophilum providing high affinity to cellulose were constructed. Content of the fused proteins in overproducing strains was 20% of total protein for CFP10-CBD and 15% for both Esat6-CBD and Ag85A-CBD. Immobilization of the fusion proteins on cellulose is a one-stage process that provides simultaneous antigen purification and absorption on cellulose. The proteins retain their antigenic properties and can be used as the components for subunit tuberculosis vaccine production or in diagnostic kits.

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.

A test system was developed to detect tuberculous infection by qualitative analysis of interferon-gamma (IFN-gamma) in the plasma samples after 20-24-hour incubation of whole blood samples in the presence of Mycobacterium tuberculosis (MBT) antigens: tuberculin PPD and a mixture of the MBT-specific recombinant antigens ESAT-6 and CFP-10. The analysis used 3 test tubes each containing 1 ml of heparinized venous blood, one of which served as a control; the other two test tubes were employed to measure antigen-induced IFN-gamma production. Whether this test system might be used to determine primary tuberculous infection was studied in 277 children and adolescents. The threshold diagnostic IFN-gamma induction level determined in the test tube containing a mixture of the antigens ESAT-6 and CFP-10 was ascertained. Postvaccine allergy was detectable if there was IFN-gamma induction in the test tube containing tuberculin and if there was no diagnostic IFN-gamma level in that containing the antigens ESAT-6 and CFP-10. The diagnostic sensitivity of detection of primary tuberculous infection was 97.6% with 94.4% specificity, which enabled this condition to be differentiated from postvaccine allergy. The level of antigen-induced IFN-gamma may be lower in relatively disseminated forms of pulmonary tuberculosis.

Bone morphogenetic protein-2 (rhBMP-2) is an osteoinductive protein factor which plays a dominant role in growth and regeneration of bone tissue. In clinical practice, bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials have not been produced yet. The fragment of the bmp-2 gene encoding a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on the basis of E. coli BL21(DE3). The level of rhBMP-2 production was approximately 25% of total cell protein. Biologically active dimeric form of rhBMP-2 was obtained as a result of isolation and purification of protein from inclusion bodies with subsequent refolding. The obtained rhBMP-2 sample contained more than 80% of the dimeric form and was able to interact with specific antibodies to BMP-2. Biological activity of the rhBMP-2 samples was verified in in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On a model of ectopic osteogenesis, it was shown that the obtained rhBMP-2 exhibited biological activity in vivo, causing tissue calcification in the site of injection. The protein activity in vivo depends on the way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The elaborated method of rhBMP-2 isolation and purification results in an increased common protein yield and ensures the maintenance of biologically active dimeric form compared to the analogues described in the literature.

Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.

Костный морфогенетический белок 2 (BMP-2) – фактор остеоиндукции, играющий основную роль в процессах роста и регенерации костной ткани. Костнопластические материалы на основе препаратов рекомбинантного белка BMP-2 человека (rhBMP-2) широко применяются в клинической практике, однако российские аналоги подобных материалов отсутствуют. Нами клонирован фрагмент гена bmp2, кодирующий зрелый белок. На основе штамма Escherichia coli BL21(DE3) создан эффективный штамм-продуцент, уровень синтеза белка rhBMP-2 в котором составляет примерно 25% суммарного белка клетки. Разработана методика выделения и очистки rhBMP-2 из телец включения с последующим рефолдингом для получения биологически активного димерного белка rhBMP-2. Получен препарат, содержащий более 80% димерной формы rhBMP-2, который взаимодействует со специфическими антителами к BMP-2. Биологическая активность препарата rhBMP-2 показана в экспериментах in vitro по индукции синтеза щелочной фосфатазы в культурах клеток С2С12 и С3Н10Т1/2. На модели эктопического остеогенеза показано, что rhBMP-2 обладает биологической активностью in vivo, вызывая образование кальцификата в области инъекции. При этом активность белка in vivo зависит от способа введения и особенностей препарата: использования раствора rhBMP-2 или препарата белка с носителем, кислого или основного буферного раствора. Предложенный способ синтеза и очистки белка rhBMP-2 позволяет повысить общий выход белка и увеличить содержание в нем биологически активной димерной формы по сравнению с известными аналогами.

The fusion gene, which consists of fragments that code the dextran-binding domain of dextraninvertase of Leuconostoc mesenteroides, subsp. mesenteroides (DBD) and human recombinant interferon-β (INF-β) with the nucleotide sequence that codes the recognition site of hydrolysis of human enteropeptidase (DDDDK) between them, was cloned in Escherichia coli cells. The effective bacterial strain that producing the chimeric DBD-INF-β protein, which consists of the IFN-β sequence, a spacer that contains ten alternate glycine and serine residues, a human enteropeptidase recognition site, and a dextran-binding domain, was constructed. Free recombinant human interferon-β was obtained as a result of the treatment of chimeric DBD-IFN-β protein and immobilized on G-25 sephadex with human enteropeptidase. The ability of free and immobilized proteins to protect human cells from viral infection was demonstrated. The developed approach can be used to produce recombinant proteins with different biological activity. Thus, these proteins can be used as a basis for the construction of new immunomodulatory and antiviral drugs, growth factors, anticancer drugs, etc.