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Biomedical subjects

A B Shevelev

Publications and source records attributed to A B Shevelev.

At least 19 recordsLinked to original sources
PubMed2006

Heterologous expression, purification, and properties of a potato protein inhibitor of serine proteinases.

The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.

Amino Acid Sequence↗
PubMed2006

[Cloning of polygalacturonase inhibitor protein genes from Solanum brevidens Fill].

New data were obtained for the Solanum brevidens Fill. nucleotide sequences coding for polygalacturonase inhibitor proteins (PGIPs), which are involved in plant defense against phytopathogenic fungi. Highly degenerate primers directed to the conserved regions of the known PGIP genes of tomato, kiwi, apple, carrot, and grape were used to clone four pgip genes and one pseudogene from the genome of S. brevidens, a species that is closely related to cultivated potato, forms no tubers, is highly resistant to phytopathogens, and is often employed in potato breeding. The sequenced part of the coding region of the new genes is 924 bp and codes for a protein of 308 amino acid residues (without the leader peptide). The genes were designated as pgipSbr1(1), pgipSbr1 (2). pgipSbr2, pgipSbr3, and pgipSbr4. The amino acid sequences of the S. brevidens PGIPs have 90.9-99.4% identity to each other and 94% identity to PGIP of Lycopersicon esculentum Mill., another member of the family Solanaceae. The amino acid residues differing between S. brevidens PGIPs were assumed to determine the selectivity of interactions with particular polyglucuronases of phytopathogenic fungi.

Amino Acid Sequence↗
PubMed2005

Molecular cloning of Kunitz-type proteinase inhibitor group B genes from potato.

Eighteen clones representing copies of four Kunitz-type proteinase inhibitor group B genes (PKPI-B) obtained by polymerase chain reaction cloning of potato (Solanum tuberosum L. cv. Istrinskii) genomic DNA were sequenced and analyzed. Three new genes were found and named PKPI-B1, PKPI-B2, and PKPI-B10: these were represented by five, two, and seven clones, respectively. The remaining four clones corresponded to the formerly characterized PKPI-B9 gene. These data show that at least four PKPI-B encoding genes are harbored in the genome of potato cv. Istrinskii. Their analysis suggests that variability of PKPI-B encoding genes in potato is limited and could be explained by cross-hybridization events in the ancestor forms rather than by random mutagenesis.

Amino Acid Sequence↗
PubMed2005

[Production of soluble form of human TNF-alpha ligand-binding domain type 1 receptor by expression in Drosophila cells].

5His-tagged human TNFalpha type I receptor (TNFR1) ligand-binding domain was produced in Drosophila cells under control of metallothionein Cu-inducible promoter and purified by Ni-NTA affinity chromatography to homogeneity. TNFR1 gene fragment was cloned by PCR from CD8+ in vitro cultured T-killer normal linage cDNA. In despite of three disulfide bonds, the recombinant protein was correctly folded which was conformed by TNFalpha ligand binding assay in ELISA variant.

Animals↗
PubMed2004

Subtilisin protein inhibitor from potato tubers.

A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (Ki = 1.67 +/- 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.

Amino Acid Sequence↗
PubMed2003

[Cloning a sequence, coding an SKT1 family protease inhibitor from potato].

Seven structurally similar clones from potato (Solanum tuberosum L.), cv. Istrinskii genomic DNA were isolated by cloning of the PCR products. It was suggested that five of these clones were the amplified copies of the same gene. Based on comparative and structural analysis of these clones, initial nucleotide structure of the gene was reconstructed. It appeared to be highly homologous (98%) to the already published sequences encoding the proteins belonging to the soybean Kunitz trypsin inhibitor family (SKTI). Comparison of the results with the previously published data on the SKTI-type proteinase inhibitors from potato of cv. Istrinskii suggests that the gene examined encodes both chains of the earlier described PSPI-21-6.3 protein [9].

Amino Acid Sequence↗
PubMed2000

Introduction of a mini-gene encoding a five-amino acid peptide confers erythromycin resistance on Bacillus subtilis and provides temporary erythromycin protection in Proteus mirabilis.

A 15-bp mini-gene was introduced into Bacillus subtilis and into stable protoplast-like L-forms of Proteus mirabilis. This mini-gene encoded the peptide MVLFV and modeled a fragment of Escherichia coli 23S rRNA responsible for E. coli erythromycin (Ery) resistance. Expression of the introduced mini-gene conferred permanent Ery resistance on B. subtilis. In L-forms of P. mirabilis, the Ery-protective effect was maintained in the course of several generations. Herewith, the mechanism of Ery resistance mediated by expression of specific short peptides was shown to exist in evolutionary distant bacteria. Three new plasmids were constructed containing the gene under study transcriptionally fused with the genes encoding glutamylendopeptidase of Bacillus licheniformis or delta-endotoxin of Bacillus thuringiensis. The Ery resistance pentapeptide (E-peptide) mini-gene served as an efficient direct transcriptional reporter and allowed to select bacillar glutamylendopeptidase with improved productivity. The mini-genes encoding E-peptides may be applied as selective markers to transform both Gram-positive and Gram-negative bacteria. The small size of the E-peptide mini-genes makes them attractive selective markers for vector construction.

Amino Acid Sequence↗
PubMed2000

Expression of bacillar glutamyl endopeptidase genes in Bacillus subtilis by a new mobilizable single-replicon vector pLF.

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease.

Amino Acid Sequence↗
PubMed1999

Propeptide of the metalloprotease of Brevibacillus brevis 7882 is a strong inhibitor of the mature enzyme.

A metalloprotease gene of Brevibacillus brevis (npr) was expressed in Escherichia coli in a soluble form as native Npr precursor. A significant fraction of the precursor was spontaneously processed, producing the N-terminal propeptide and the mature enzyme. A strong inhibition of the mature Npr by its own propeptide in the crude lysate was observed even in the absence of the covalent linkage between them. Pure precursor, propeptide and the mature Npr were isolated and kinetic parameters of the mature enzyme inhibition by the propeptide were determined. The inhibition is of the tight-binding competitive type with Ki 0.17 nM. Inhibition of metalloproteases from Brevibacillus megaterium and thermolysine by the heterologous propeptide of the Npr from B. brevis was much weaker or none.

Amino Acid Sequence↗
PubMed1999

Bacillus intermedius glutamyl endopeptidase. Molecular cloning and nucleotide sequence of the structural gene.

The glutamyl endopeptidase gene of Bacillus intermedius was cloned from a genomic library expressed in Bacillus subtilis and sequenced (EMBL accession number Y15136). The encoded preproenzyme contains 303 amino acid residues; the mature 23-kDa enzyme consists of 215 residues. The mature enzyme reveals 38% of identical residues when aligned with the glutamyl endopeptidase from Bacillus licheniformis, whereas only five invariant residues were found among all known glutamyl endopeptidases. The amino acid residues that form the catalytic triad (H47, D98, and S171) as well as H186 participating in the binding of the substrate carboxyl group were identified. It seems that the structural elements responsible for the function of glutamyl endopeptidases from various sources are highly variable.

Amino Acid Sequence↗
PubMed1998

Expression of the carboxypeptidase T gene from Thermoactinomyces vulgaris in stable protoplast type L-forms of Proteus mirabilis.

The structural gene of the carboxypeptidase T (cpt) was successfully expressed in cell wall-less L-form cells of Proteus mirabilis. The DNA sequence encoding the PhoA leader peptide was fused with a truncated cpt gene encoding the mature enzyme. The modified gene in a pUC-based kanamycin resistance vector under the control of the lac promoter was transformed into L-form cells of P. mirabilis. They were able to produce the recombinant CpT both as a secretory and as a cell-bound insoluble form. The co-secretory processing of the PhoA leader peptide was quite efficient. The yield of the secreted CpT was not less than 20 mg l-1 and should be improvable.

Amino Acid Sequence↗
PubMed1998

A new PCR-based approach to a fast search of a wide spectrum of cry genes from Bacillus thuringiensis strains.

A pair of highly degenerated primers was adapted to carry out a single-step PCR-detection of any known and probably unknown cry genes of classes cry1, cry4 and cry9 encoding for 130 kDa protein delta-endotoxins in the natural Bacillus thuringiensis (BT) strains. The Southern hybridization of the product has demonstrated that essentially remote cry-genes like cry1Aa and cry9A (cryIG) could be represented in the single amplificate if they are simultaneously present in the genome of the analyzed strain. Four genes were detected by the proposed scheme in the BT ssp. galleriae 11-67. One of them, gene cry1Ga1 was originally found and cloned using the PCR-amplification product obtained from the genomic DNA of this strain as a probe. The new gene was completely identical to one cloned by B. Lambert (unpublished, EMBL accession number Z22510) and essentially related to cryIM (EMBL accession number Y09326), renamed according to the new nomenclature as cry1Ga2.

Bacillus thuringiensis↗
PubMed1998

Purification and characterization of P33 protein of Candida utilis homologous to bgl2p of Saccharomyces cerevisiae; comparative analysis of the role of these proteins in molecular organization of the yeast cell walls.

P33 protein was isolated from the cell walls of Candida utilis. Homology between P33 and Bgl2p proteins from the cell walls of Saccharomyces cerevisiae was shown. The important role of these proteins in molecular organization of yeast cell walls was demonstrated using trypsin proteolysis and the "gene disruption" method.

Candida↗
PubMed1997

A novel delta-endotoxin gene cryIM from Bacillus thuringiensis ssp. wuhanensis.

A new cryI-related sequence designated cryIM was cloned using an immunoscreening technique from ssp. wuhanensis of Bacillus thuringiensis (BT), previously reported to produce multiple Cry proteins [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034]. Analysis of the cryIM nucleotide sequence revealed an ORF, BTII-type promoter-like sequence, peculiar for such genes, a translation initiation element and a putative transcription terminator. Nevertheless, its product was not previously found in the crystals of the host strain [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034] which shows its weak or absent natural expression. The amino acid sequence of 1151 residues encoded by the continuous reading frame of cryIM is not identical but is essentially similar to the other delta-endotoxins of the CryI class. An IS231-like sequence was found 400 bp downstream of the cryIM stop codon and a fragment of the cryIAb gene was located 3 kb upstream of its initiator codon in the same orientation. Artificial expression of the cloned gene in E. coli under the control of the lacZ promoter allowed us to obtain its hypothetical protein product.

Amino Acid Sequence↗
PubMed1993

Primary structure of cryX**, the novel delta-endotoxin-related gene from Bacillus thuringiensis spp. galleriae.

A cry-related sequence, designated cryX (EMBL X75019), was localized upstream of cryIG, the delta-endotoxin gene cloned from spp. galleriae of Bacillus thuringiensis and sequenced earlier [(1991) FEBS Lett. 293, 25-28]. Analysis of the cryX complete nucleotide sequence enabled us to explain its virtual crypticity and to reveal the chimeric structure of the genes, cryX and cryIG. The amino acid sequence of 1,151 residues encoded by the continuous reading frame of cryX is similar to the other delta-endotoxins but differs essentially from them.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of a novel delta-endotoxin gene cryIg of Bacillus thuringiensis ssp. galleriae.

A gene cryIg coding for entomocidal protein delta-endotoxin of Bacillus thuringiensis ssp. galleriae str. 11-67 named CryIG has been cloned and sequenced (EMBL accession number X58120). The deduced amino acid sequence that contains 1156 amino acid residues shows only 28% of identical residues, when compared with other delta-endotoxins of the CryI family. The extent of identity is substantially higher for some regions of the sequence ('conserved blocks'), that presumably bear important structural or functional properties. This implies that CryIG delta-endotoxin follows the same type of polypeptide chain folding as other CryI proteins, whereas peculiarities of primary structure help to explain its unique specificity.

Amino Acid Sequence↗