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M A El'darov

Publications and source records attributed to M A El'darov.

At least 19 recordsLinked to original sources
PubMed1998

[Isolation and specificity of novel restriction endonucleases Bsp40091 and AsiI, isoschizomers of BamHI].

New type II restriction endonucleases AsiI and Bsp40091 are detected in Azotobacter species N55 and Bacillus species 4009, respectively. Purified preparations of the restriction enzymes free from interfering nucleases and phosphatases were obtained by column chromatography on phosphocellulose and heparin-sepharose (Asil) and phosphocellulose and DEAE-cellulose (Bsp40091). The yield of purified AsiI and Bsp40091 was 16 x 10(3) and 8 x 10(3) units per g of wet cells, respectively. The above restriction endonucleases recognize the 5'-G decreases GATCC-3' sequence on double-stranded DNA and cleave it as shown, thus being true isoschizomers of BamHI restriction endonuclease.

Azotobacter↗
PubMed1998

[Novel site-specific endonucleases from Brevibacterium species].

New site-specific endonucleases BecAI and BecAII have been detected in Brevibacterium species A. Endonuclease BecAII free from contaminating nonspecific endonucleases, exonucleases, and phosphatases was isolated by column chromatography on phosphocellulose, heparin sepharose, and DNA cellulose. It recognizes and cleaves the 5'-GG decreases CC-3' sequence and is a true isoschizomer of HaeIII restriction enzyme. The other restriction endonuclease, BecAI, cleaves Ad2 DNA at least by 2 sites but not the DNA of phage lambda, T7, SV40, phiX174, and plasmides pBR322 and pUC19. The substrate specificity of BecAI indicates its appurtenance to the super rare restriction endonucleases.

Brevibacterium↗
PubMed1996

[Site-specific BcuAI endonuclease from Bacillus cereus A].

A new restriction endonuclease was isolated from the Bacillus cereus BKM B-814 by means of the cell disruption with ultrasonication, ammonium sulfate fractionation of the cell-free extract, and chromatography on DEAE-Sepharose to give about 1400 U of the enzyme per gram of cells. The enzyme revealed the maximum activity at 30-37 degrees C, pH 7.6-8.2, and 5-10 mM MgCl2 under a high ionic strength (50 mM Tris-HCl, 100 mM NaCl). The site-specific endonuclease BcuAI was found to recognize the 5' G decreases G(A/T)CC sequence in double-stranded DNA and cleave it as shown with the arrow, thus being a true isoschisomer of the AvaII restriction endonuclease.

Bacillus cereus↗
PubMed1994

[Isolation, purification, and characteristics of the new restriction enzymes BciBI and BciBII, produced by Bacillus circulans].

New site-specific endonucleases BciBI and BciBII have been detected in Bacillus circulans. The enzymes were purified by fractionation of cell-free extract with polyethylene imine and ammonium sulphate (40-80% of saturation) followed by chromatography on DEAE-sepharose, blue-sepharose and phosphocellulose. The endonucleases BciBI and BciBII were separated only at the final step of the purification--by chromatography on the phosphocellulose column. The yields of BciBI and BciBII were 600 and 10,000 U/g of cells. It was found that restriction endonucleases BciBI and BciBII are isoschizomers of ClaI and BstNI, respectively.

Bacillus↗
PubMed1994

[A prosegment of the yeast alpha-factor controls a heterologous protein (human growth factor) in Saccharomyces cerevisiae culture media].

The role of the yeast pheromone prosegment--alpha-factor--in the export of the heterologous protein, the human growth hormone (hGH), in the culture medium of S. cerevisiae has been studied. Using genetic engineering constructions, it has been shown that different N-terminal signal peptides (SP) are not able to provide the hGH export. Transformant cells carrying the plasmid with a hybrid sequence encoding the prosegment-hGH (without SP) accumulate non-glycosylated pro-hGH in the cytosol. Only the combination of SP and the prosegment as the N-terminal fragment of the hGH precursor results in the processing and export of the mature form of the hormone. The origin (or type) of SP is of no significance. The glycosylation inhibitors--2-deoxy-D-glucose and tunicamycin--suppress the export but not the entry of hGH into the periplasm, thus indicating a critical role of the intactness of the prosegment polymannose chains for the efficient export of heterologous protein. A conclusion is drawn that the two preprosegment parts play different roles. The SP constituent of prepro-hGH introduces pro-GH into the general secretory pathway, while the prosegment resulting from the SP cleavage directs hGH from the cell into the culture medium.

Amino Acid Sequence↗
PubMed1992

[Specific endonuclease BbvBI from Bacillus brevis].

A new restriction endonuclease BbvBI free from contaminating nonspecific nucleases and phosphatases was isolated from the Bacillus brevis cells. The enzyme was purified by fractionating the sonicated cell-free extract in a two-phase PEG/dextran system and subsequent chromatographies on DEAE-sepharose, blue sepharose and heparin sepharose. The endonuclease BbvBI displayed the maximal activity at 45 degrees C, pH between 8.0 and 8.5, MgCl2 concentration in the range of 5-10 mM and at the low ionic strength. It is shown that the enzyme cleaves the sequence G'GYPC'C, with the preferential cleavage of GGTACC and GGCACC sites as compared with GGTGCC and GGCGCC. Thus, the restriction endonuclease BbvBI is a true isoschizomer of nuclease BanI.

Bacillus↗
PubMed1991

[BsoAI--a new site specific endonuclease from Bacillus coagulans].

A new site-specific endonuclease was detected in toluene lysates of Bacillus coagulans AUCM B-732 and designated as BcoAI. The enzyme was purified by fractionation of the cell-free extract in the two-phase PEG/dextran system followed by chromatography on DEAE-sepharose and phosphocellulose and shown to be free of nonspecific nucleases and phosphatases. BcoAI has three cleavage sites on lambda DNA, but does not cleave SV40, pBR322 and pUC19 DNA. BcoAI recognizes the sequence 5' CAC decreases GTG 3' on double-stranded DNA and cleaves it as indicated by the arrow to yield blunt-ended DNA fragments. Thus, BcoAI is a true isoschizomer of PmaCI from Pseudomonas maltophila C.

Autoradiography↗
PubMed1991

[Localization of acid phosphatase in Saccharomyces cerevisiae and its export into culture media depends on the type of the N-terminal signal peptide].

The aim of this work was to study the character of intracellular distribution and efficiency of yeast acid phosphatase export depending on the type of the N-terminal signal peptide used. A number of plasmids carrying the acid phosphatase genes with different signal peptides sequences was constructed. The main site of the enzyme accumulation for the variant containing its own acid phosphatase signal peptide was the periplasm. Approximately the same pattern was observed when the hybrid signal peptide consisting of acid phosphatase signal peptide and alpha-factor preprosegment tandem was used. Unlike the above-mentioned systems the strain carrying acid phosphatase under the control of alpha factor preprosegment was able to export the enzyme into the culture medium. The experiments have shown the possibility of changing the final localization of secretory proteins by replacing the N-terminal signal peptide.

Acid Phosphatase↗
PubMed1988

[Behavior in a cell of artificial mini-chromosomes during induced transcription of centromeric DNA].

Hybrid yeast plasmids were constructed, containing the centromere loci CEN3 under the control of two inducible yeast promoters--GAL10 and PHO5. It was shown, that during the induction of transcription from the GAL10 promoter the decrease in mitotic stability of minichromosome is affected both by partial disruption of centromere function by transcription and by influence of galactose on the number of residual cell divisions. In two strains the activity of GAL10 promoter was considerably higher than that of the PHO5 promoter. It is proposed to use the effect of minichromosome destabilization for evaluation of the relative promoter strength.

Centromere↗
PubMed1987

[Expression of a gene for hybrid protein containing the [Val8] amino acid sequence of calcitonin in yeast cells].

A recombinant plasmid has been constructed, which directs the synthesis of a hybrid protein, yeast repressible acid phosphatase [Val8]calcitonin, in yeast. The plasmid contains a truncated gene (pho5) acid phosphatase lacking 96 C-terminal amino acids replaced by the synthetic gene for human calcitonin and sequences required for the plasmid propagation in transformed yeast cells. A modified RIA method using immobilisation of protein extracts on solid supports was developed to monitor the expression of the hybrid protein. By use of this method, as well as by standard RIA of CNBr-cleaved protein extracts, synthesis of a calcitonin-related protein was detected in extracts of transformed strains grown under conditions inducing pho5 promoter.

Amino Acid Sequence↗
PubMedDate not supplied

[Expression constructs of inducible isoform of nitric oxide synthase in Escherichia coli cells].

In the present work we describe the construction of expression system for inducible murine macrophage nitric oxide synthase (iNOS) in E.coli. For this purpose a framework of translation iNOS was cloned in the expression vector pCWori +. As biosynthesis of active iNOS requires coexpression of calmodulin (CaM), for obtaining functional expression of this protein we conducted amplification of an appropriate site of the library total cDNA a frog Xenopus laevis, then plasmids for coexpression of calmodulin were constructed under a control tac and T7 promotors. Recombinant iNOS was functionally active as revealed by the analysis of CO-reduced spectrums, detection of derivation NO with the help of reaction conversion HbO2 in metHb, and also identification of a molecule NO by EPR method. The output of recombinant iNOS at usage of different constructions varied from 10 up to 22 mg/l culture, and specific activity was from 0.42 up to 0.64 U/mg of protein. These data coincide with the earlier published results of other investigators. It was established, that the expressed iNOS is associated to a membrane fraction of cells, thus in the 105,000 g-supernatant the activity of an enzyme is not detected. The data on membrane localization iNOS are inconsistent with general notion this enzyme is soluble.

Animals↗
PubMedDate not supplied

[Heterologous expression of eukaryotic CYP450. 1. Heterologous expression of cytochrome P450 2B4 using groups with various affinity in E. coli].

The expression levels of cytochrome P450 2B4 variants with N- and C-terminal modifications were compared and some of the enzymatic characteristics of recombinant proteins studied. Following C-terminal hybrids for CYP2B4 gene were constructed: 1) with intein-chitin binding domain cassette 2) with hexahistidine tag. These modifications were combined with P450 2B4 glutathione-S-transferase N-terminal fusions [Pernecky S.J., et. al., (1995) Arch. Biochem. Biophys., 318, 446-456]. The obtained constructs provided for the synthesis of full-length protein products in E. coli cells with holoenzyme yield at the levels of 200-1000 nmoles/l of the bacterial culture. Partial in vivo proteolysis was observed for C-terminal fusions with intein moiety despite the presence of glycine aminoacid residue at the junction of two proteins. The principle inapplicability of standard purification scheme for isolation of P450 2B4-intein fusions is demonstrated, since the P450 domain is inactivated at 40 mM DTT concentrations. The recombinant full-length CYP 2B4 with C-terminal oligohistidine tail was expressed under the control of T7 promoter and purified using immobilized metal-ion chelating chromatography. The C-terminal hexahistidine tag does not affect the catalytic properties of recombinant enzyme in 7-pentoxyresorufin O-dealkylation reaction.

Aryl Hydrocarbon Hydroxylases↗
PubMedDate not supplied

[Cloning of the human cytochrome p450 2B6 gene in Escherichia coli cells].

Human cytochrome P450 2B6 gene from plasmid pUC9, carrying cytochrome CYP2B6 cDNA was cloned into eucariotic expression vector pcDNA 3.1 (+). Cytochrome P450 2B6 gene in recombinant plasmid pcDNA 3.1 (+)/CYP 2B6 was expressed in E. coli cells. The expression of catalytically active recombinant protein was 60-100 nm per liter of the culture medium.

Aryl Hydrocarbon Hydroxylases↗