Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album.

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that - at a subsequent stage of transfection into yeast cells - it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein's secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 µg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

Correction to: Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells.

The original publication has been updated. The acknowledgment was omitted from the original article and is published below.

Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells.

Non-glycosylated, recombinant human granulocyte colony-stimulating factor (rhG-CSF), produced by Escherichia coli (filgrastim, leukostim) is widely used to treat a number of serious human diseases and aids in the recovery post bone marrow transplantation. Although glycosylation is not required for the manifestation of the biological activity of G-CSF, a number of studies have shown that the carbohydrate residue significantly increases the physicochemical stability of the G-CSF molecule. Therefore, the aim of the present study was to design a Pichia pastoris strain capable of producing glycosylated rhG-CSF, and to study its effects on rat bone marrow cells. The nucleotide sequence of the rhG-CSF gene has been optimized for expression in P. pastoris, synthesized, cloned into the pPICZαA vector and expressed under the control of the AOX promoter in P. pastoris X33. One of the selected clones secreting rhG-CSF, produced 100-120 mg/l of rhG-CSF three days post-induction with methanol. The recombinant cytokine was purified using two-step, ion-exchange chromatography. The final yield of purified G-CSF was 35 mg/L of culture medium. The biological activity of rhG-CSF was examined in rat bone marrow cells. The P. pastoris strain was designed to produce relatively high levels of rhG-CSF. The rhG-CSF protein had a strong stimulating effect on the growth of rat bone marrow cells, which was comparable to that of the commercial drug leukostim, but showed a more persistent effect on granulocyte cells and monocyte sprouts, enabling the enhanced maintenance of the viability of the cells into the 4th day of incubation.

[Effect of a gel based on recombinant human angiogenin on the healing of donor palate wounds]. / Vliianie gelia na osnove rekombinantnogo angiogenina cheloveka na zazhivlenie donorskikh ran tverdogo neba.

The aim of the study was to study the effect of the gel on the basis of recombinant human angiogenin on the rate of regeneration of donor palatal wounds. The study involved 20 patients (8 men and 12 women) aged 32 to 55 years. Patients were divided into two groups: the 1st group is a study group (n=10), whose patients in the postoperative period used a gel based on recombinant human angiogenin, the 2nd group is a control group (n=10) in which a gel based on recombinant human angiogenin was not used. Patients in both study groups underwent vestibuloplasty with simultaneous plasty of the attached keratinized gingiva with a free gingival graft from the area of the hard palate. The operations were carried out at the stage of disclosing dental implants, simultaneously with the installation of healing abatements or 4 weeks before dental implantation. For histological examination, tissue samples were obtained from the region of the edge of the donor's wounds of the palate at the 7th and 14th days after surgery. As a result of the study, significant differences were found in the comparison groups when assessing the processes of inflammation, angiogenesis and epithelization. The local application of the gel containing recombinant human angiogenin resulted in a rapid decrease in the intensity of inflammation in lamina propria mucosae and a significant decrease in the bulk density of cell infiltrates, accelerating regeneration. This is primarily due to the stimulation of the development of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and increased blood supply to the affected area, as well as an increase in the proportion of fibroblasts. The most important observation was the increase in the rate of epithelialization of donor wounds of the hard palate.

[IMMUNOCHEMICAL ANALYSIS OF RECOMBINANT CHIMERIC POLYPEPTIDE OspC(gar+afz) OF BORRELIA GARINIIANIP B. AFZELI ISOLATES].

AIM: Comparative study of antigenic properties of recombinant proteins OsPCgar and OsPCafz and recombinant chimeric polypeptide OspCgar+afrz, that contains amino acid sequences of mature immune dominant OspC proteins of West-Siberian isolates of Borrelia garinii (OspCgar) and B. afzelii (OspCafz), and evaluation of possibility of their use as antigens during creation of test-systems for serodiagnostics of Lyme borreliosis (LB) on the territory of Western Siberia. MATERIALS AND METHODS: Recombinant chimeric polypeptide OSpCgar+af, and recombinant mature proteins OSPCgar and OspCafz, obtained by expression of the corresponding genes in Escherichia coli cells; purified by affinity chromatography in Ni-NTA-sepharose CL-6B and studied by EIA method for the ability to bind antibodies from sera of LB patients. RESULTS: A difference in sensitiv- ity of determination by EIA method of specific IgM and IgG against borreliae in blood sera of LB patients with localized stage of the disease during use of OspCgar,'OSPCafz and OspCgar+afz chimera as antigens was shown. Chimeric antigen OSPCgar+afz was established to show higher antigenic activity compared with each of the OspCgar or OSPCafz antigens separately. CONCLUSION: The results of the study allow to examine.the recombinant chimeric polypeptide OspCgar+afz as a pos- sible component during creation of test-systems for serodiagnostics of LB on the territory of West, Siberia.

[Evaluation of significance of Borrelia garinii spirochete recombinant protein DbpB for serodiagnostics of ixodes tick-borne borreliosis].

AIM: Obtaining recombinant protein DbpB of West Siberia Borrelia garinii 20047 isolate and evaluation of its antigen activity for the possible use in serodiagnostics of ixodes tick-borne borreliosis (ITB). MATERIALS AND METHODS: Coding region of dbpB gene of novosibirsk B. garinii 20047 isolate was amplified by PCR and cloned as part of expressing pETm vector in Escherichia coli BL21 (DE3) strain cells. Recombinant protein DbpB produced by the selected clone was studied by EIA method for its ability to react with sera antibodies of ITB patients. RESULTS: E. coli BL21 (DE3) clone producing recombinant protein DbpB in quantity of 30% of total E. coli cell protein was obtained. Homology of amino acid sequence of recombinant protein DbpB of novosibirsk B. garinii 20047 isolate with primary structures of B. garinii, B. afzelii and B. burgdorferi sensu stricto spirochete genospieces DbpB proteins presented in GenBank database was 98.4, 77 and 73%, respectively. Sensitivity of immune enzyme detection in sera of ITB patients with migrating erythema of IgM and IgG reacting with DbpB antigen was 13.9 and 20.0%, respectively. Frequency of detection of IgM and IgG against DbpB in patient sera with disseminated ITB form was 15.7 and 43.8%, respectively. Specificity of immune enzyme detection of antibodies against recombinant antigen DbpB in which sera of syphilis, rheumatoid arthritis patients and healthy donors used as control sera was 100%. CONCLUSION: DbpB recombinant protein of novosibirsk B. garinii 20047 isolate may be used as one of antigens for highly specific serodiagnostics of ITB disseminated stage.

[Recombinant strain producing thermostable lipase from Thermomyces lanuginosus immobilized into nanocarbon silica matrices and properties of the prepared biocatalyzers].

Multicomponent composite biocatalyzers with lipolytic activity have been studied. These biocatalyzers were prepared through the immobilization of a recombinant producer strain of thermostable lipase from Thermomyces lanuginosus into SiO2 xerogel, which contains a nanocarbon component, i.e., multilayered carbon nanotubes with varying diameters, and also bulblike structured carbon nanospheres ("nanobulb"). The properties of lipase were studied both in cell suspensions of a recombinant producer strain constructed based on E. coli BL21(DE3) and in the immobilized state with regard to the structure and dispersibility of the nanocarbon component used in the composition of the biocatalyzers. It was shown that the recombinant intracellular lipase exerted its activity in a reaction of tributirin hydrolysis on average comprising 50 U/mg of dried cells and had a high level of thermostability. Upon heating in olive oil at 100 degrees C, the inactivation constant and the period of semi-inactivation comprised 6 x 10(-3) min(-1) and 2 h, respectively, exceeding by one order the thermostability of lipase in a buffer solution. Biocatalyzers that contained aggregated "thick" nanotubes with a diameter of 20-22 nm had the maximum initial activity-250 U/g.

[Immunochemical analysis of recombinant protein FlaA of Western Siberian Borrelia garinii isolate].

AIM: Study of the ability of Western Siberian Borrelia garinii 20047 isolate recombinant protein FlaA to react with sera antibodies of ixodes tick borreliosis patients. MATERIALS AND METHODS: Recombinant antigen FlaA, sera of ixodes tick borreliosis patients, genetic engineering methods, solid phase EIA, and parametric and nonparametric statistical methods were used in the study. RESULTS: Recombinant form of mature flagellar protein FlaA of B. garinii 20047 was obtained. In EIA study of sera of ixodes tick borreliosis patients with migrating erythema and without it, IgM or IgG against FlaA antigen were detected in more than 30% of sera. Indicator of the detection of IgM against FlaA antigen in sera of ixodes tick borreliosis patients with migrating erythema and without it was 43.3% and 33.3% respectively. CONCLUSION: The results obtained show a significant antigenic activity of recombinant protein FlaA of Western Siberian B. garinii isolate and the perspectives of its use for serodiagnostics of ixodes tick borreliosis.

[Enzyme immunoassay-based analysis of OspC recombinant proteins from Borrelia garinii and Borrelia afzelii isolated in West Siberia].

AIM: To study the ability of OspC recombinant proteins from Borrelia garinii and Borrelia afzelii isolated in West Siberia to interact with serum antibodies from patients with tick-borne borreliosis (TBB). MATERIALS AND METHODS: Recombinant antigens OspC B. garinii and OspC B. afrelii, serum samples from patients with TBB were used as well as solid-phase enzyme immunoassay and parametric and non-parametric statistical methods. RESULTS: Higher antigenic activity of B. garinii OspC compared with OspC from B. afzelii was observed when these recombinant proteins were compared in enzyme immunoassay. Detection rate of class M and G immunoglobulins to B. garinii OspC in sera of patients with TBB was 60.5% and 70% respectively. CONCLUSION: Obtained results indicate high immunoreactivity of OspC recombinant proteins from B. garinii and B. afzelii and point to perspective of their combined use for serological diagnostics of TBB.

[Isolation of the recombinant proteins OspC and the fragment FlaA (F-FLAA) from the western-Siberian Borrelia garinii NT29 isolates and the study of their immunochemical properties].

The structural proteins OspC and FlaA of the Lyme disease (LD) agent are known to be the basic antigens, which induce the humoral immune response at the initial stage of the disease. The goal of this work was to obtain the recombinant OspC and a fragment of the FlaA protein (f-FlaA) from the Western Siberian Borrelia garinii NT29 isolates and to assess the possibility of their use for the LD diagnosis. Encoding regions of the OspC and f-FlaB genes were amplified using PCR inserted in the pREB expressive vectors and cloned in the E. coli str. BL21 and C-600, respectively. The recombinant OspC and f-FlaA proteins were purified using affinity chromatography on Ni-NTA-sepharose 6A, and their ability to bind serum antibody of patients with Lyme disease was tested using western-blot and ELISA methods. The results of the analyses suggest that these proteins can be considered as promising components for elaboration of diagnostic tests for LD. The prototype of the ELISA diagnostic test was designed on the basis of the OspC and f-FlaA recombinant antigens. This test provides satisfactory parameters of diagnostic specificity (70.0%) and sensitivity (85.0%).

Development of a biosensor test system with GFP reporter protein for detection of DNA damages.

A sensitive biosensor test system was developed for evaluation of premutation effects of propellants on the cell genome.

[The 2003 results of monitoring of influenza A virus in the populations of wild birds in the south of Western Siberia].

The paper presents the results of isolation of influenza A virus from 97 cloacal swabs of 11 species of aquatic and semiaquatic wild birds collected on the Chany Lake (the south of Western Siberia, Ob-Irtysh interarea). Six strains with subtypes H2 (2 strains), H3 (3 strains), and H5 (1 strain) were isolated from mallard ducks (Anas platyrhynchos). The total infection rate in the examined birds was 6.2% and that in the ducks was 9.7%. The paper deals with the phylogenetic analysis of hemagglutinin of genes of isolates and with the comparison of the obtained results with the 2002 data in the same region. Analysis of H5 strain hemagglutinin proteolytic site permits one to regard this strain as non-pathogenic.

[Genetic variety of influenza A virus in the populations of wild birds in the south of Western Siberia].

Influenza A virus variants belonging to H3 and H4 subtypes were isolated from wild ducks inhabiting in the south of Western Siberia. Phylogenetic analysis of hemagglutinin (HA) gene of these viruses has revealed that H3 isolates are closely related to those isolated from the bird inhabiting in West Europe (A/Teal/Germany/wv01r/01, A/Duck/Ukraine/1/63) and China (A/Aquatic bird/Hong Kong/399/99); and those isolated from the birds inhabiting in Germany (A/Garganey/Germany/wv157k/01, A/Teal/Germany/wv153k/01). Thus, closely related influenza A virus variants circulate in the populations of the wild birds inhabiting in greatly spaced regions of Eurasia.

[Mycobacterium tuberculosis detection by polymerase chain reaction and identification of M. tuberculosis strain]. / Vyiavlenie mikobakterii tuberkuleznogo compleksa metodom polimeraznoi tsepnoi reaktsii s odnovremennoi identifikatsiei vida M. tuberculosis.

A rapid multiprimer PCR method for detection of Mycobacterium tuberculosis complex (MTC) and simultaneous identification of M. tuberculosis in clinical samples has been developed. The method is based on simultaneous amplification of two targets: a 401 bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 544 bp fragment from the RD1 genome region which is specific for MTC but absent in BCG strains. Polymerase inhibitors in this study were detected by internal control in each test. Detection sensitivity was 25 copies of M. tuberculosis genomic DNA. Seven methods for isolation of mycobacterial DNA were compared and the technique with chloroform extraction was selected as the most efficient. The proposed method was used for analysis of 37 clinical samples and the results were compared with the results of culturing, acid-fast bacilli staining, and clinical diagnosis. The method proved to be sufficiently sensitive and specific for detection of mycobacterial DNA. Moreover, in countries with only two main pathogenic species of MTC circulating (M. tuberculosis and M. bovis) this method can be used for differentiation of these two species.

Dynamic study of HBsAg and HBeAg in saliva samples from patients with hepatitis B infection: diagnostic and epidemiological significance.

Analysis of 505 cases history of patients among men with viral hepatitis demonstrates that HBV infected patients represent 68.9% of the total and that a non-parenteral rate of transmission is the most likely means of hepatitis B infection. Saliva and serum testing for the presence of specific HBV markers (HBsAg, HBeAg and HBV DNA) at different phases of the infection process were carried out to review the diagnostic and epidemiological value of saliva samples from patients with acute viral hepatitis B. The frequency of HBsAg detection by Enzyme Immune Assay (EIA) in saliva of patients in acute period was found to correlate with the frequency of its detection in serum. In early convalescence the frequency of detection of that antigen in serum (59.5% of patients) was significantly higher than in saliva (23.8%) (P < 0.001). The frequencies of HBeAg detection by EIA in saliva samples was significantly higher than that in serum samples in both acute phase (84.3% and 28.1% of patients, respectively) and in early convalescence (56.2% and 3.1% of patients, respectively). The study of frequencies of detection of these antigens in the dynamics of the disease up to the total recovery of patients (observations were carried out for the period of 60 days and longer) showed that in most patients there was a faster disappearance HBsAg from saliva than from serum. By the end of second month this antigen was detected in saliva of only 8.3% of patients whereas in serum in the same period HBsAg was detected in 33.3% of patients. HBeAg became undetectable in blood whereas HBs-antigenemia was still pronounced, and a month after the beginning of the disease it was not found in serum specimens. In saliva, HBeAg was detected in 95.8% of patients observed directly after admission. A month after the beginning of the disease it was detected in saliva of 66.7% of patients and, by the end of observation period, in 12.5% of patients recovered from viral hepatitis. HBV DNA revealed by PCR in saliva and serum of HBV-infected patients was detected in acute period not only in serum (84.6% of cases) but also in saliva (46.2% of cases). The data illustrate the diagnostic value of saliva and point to the possible role of saliva as a source of HBV infection.

[Protective activity of vaccinia virus envelope proteins isolated using nonionic detergents]. / Protektivnaia aktivnost' belkov obolochki virusa ospovaktsiny, vydelennykh s ispol'zovaniem neionnykh detergentov.

Several detergents and chemical compounds--Tweens 40, 60, 80, Triton X-100, Triton WR-1340, polyvinylpyrrolidone, dimethylsulfoxide, urea, n-butyl, MESK, and combinations thereof were used for the isolation of surface proteins of vaccinia virus. Optimal conditions for the treatment of the virus with detergents were selected, permitting isolation of vaccinia virus surface proteins p35 and p61. Mouse experiments yielded data on the protective properties of the isolated proteins. Protein p35 may turn to be one of the major proteins responsible for the formation of protective immunity in vaccination with vaccinia virus.

[An analysis of the amino acid sequence of the heavy subunit of the hemagglutinin in influenza virus A/Alma-Ata/1417/84]. / Analiz aminokislotnoi posledovatel'nosti tiazheloi sub''edinitsy gemaggliutinina virusa grippa A/Ama-Ata/1417/84.

An analysis of the amino acid sequence of influenza A/Alma-Ata/1417/84 (H1N1-Hsw1N1 serovariant) virus hemagglutinin heavy chain deduced from the nucleotide sequence of cloned full-size DNA complementary to the 4th segment of genome RNA was carried out. Unlike A/New Jersey/8/76 virus, the hemagglutinin of the virus under study was found to be more similar in the rate of HA1 homology, amino acid sequence of the signal peptide, antigenic sites Sa, Ca, and the receptor-binding site to human influenza viruses isolated in the 1930-1980-ies, in particular to influenza A/Taiwan/1/86 virus. It is assumed that an influenza virus more adapted to the human population like the strain A/Alma-Ata/1417/84 may be an etiological factor of a new influenza pandemic.

[Synthesis, cloning, and determination of the primary structure of a full-length DNA copy of the gene for influenza A/Alma-Ata/1417/84 virus (H1N1-serovariant HSW1N1) hemagglutinin]. / Sintez, klonirovanie i opredelenie pervichnoi struktury polnorazmernoi DNK-kopii gena gemaggliutinina virusa grippa A/Alma-Ata/1417/84 (N1N1-serovariant HSW1N1).

The full-length copy of the hemagglutinin gene RNA of the influenza virus A/Alma Ata/1417/84 (Hsw1 N1-serovariant) has been synthesized and cloned on Escherichia coli plasmid pBR327. The complete nucleotide sequence of the cloned DNA copy was determined by the Maxam-Gilbert procedure. The predicted amino acid, sequence of HA1 hemagglutinin subunit was compared with the sequences of HA1 subunits from other H1N1-subtype influenza virus strains. It has been found that the structure of the HA1-subunit of the studied strain is most similar to the structure of the identical region of the A/New Jersey/18/76 hemagglutinin.

[Production of monoclonal antibodies to staphylococcal alpha-toxin using the technique of in vitro immunization]. / Poluchenie monoklonal'nykh antitel k stafilokokkovomu alfa-toksinu pri ispol'zovanii tekhniki immunizatsii in vitro.

The aim of this study is production of monoclonal antibodies (MAB) to staphylococcal alpha-toxin (SAT). SAT was obtained from culture medium of S. aureus s. 0-15 with ion-exchange chromatography and chromatofocusing. SAT was conjugated with CH-sepharose 4B and used for in vitro immunization of spleen cells, extracted from intact BALB/c mouse.

[The protein-synthesizing apparatus of a BHK-21 (C-13) cell culture and of its large-cell clone C-9]. / Issledovanie beloksinteziruiushchego apparata kul'tury kletok VNK-21 (C-13) i ee krupnokletochnogo klona C-9.

Cell line BHK-21 (C-13) and its large cell clone C-9 differ in morphology, karyotype and cultural properties. Clone C-9 is polyploid. It has been shown that C-9 clone cells display 2.5-3-fold excess in the nucleolus organizer region (NOR) in chromosomes, and 2-3 times higher intensity of protein synthesis and of ribosomal material content compared to the original line. Data of sedimentation analysis and of protein synthesis activity of the total ribosomal material in the cell-free translational system from rabbit reticulocytes allow to conclude that the quantity and size of polyribosomes in C-9 cells are higher than in cells of the original line. Apparently, the quantity of NOR-chromosomes reflect the activity of protein translation system of investigated cells.