[Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

[Production of humanized F(ab')2 fragment of rabies blocking antibodies in Pichia pastoris yeast].

The yeast strain Pichia pastoris, a producer of humanized F(ab')2 fragments of rabies-blocking antibodies, has been obtained. Human chaperone BiP coexpression caused a twofold increase of the immunoglobulins secretion level. The use of Fos and Jun zippers in the composition of heavy chains facilitated the dimerization of F(ab')2 fragments of the shared pool of secreted immunoglobulins up to 75%.

[Distiller Yeasts Producing Antibacterial Peptides].

A new method of controlling lactic acid bacteria contamination was developed with the use of recombinant Saccharomyces cerevisiae strains producing antibacterial peptides. Genes encoding the antibacterial peptides pediocin and plantaricin with codons preferable for S. cerevisiae were synthesized, and a system was constructed for their secretory expression. Recombinant S. cerevisiae strains producing antibacterial peptides effectively inhibit the growth of Lactobacillus sakei, Pediacoccus pentasaceus, Pediacoccus acidilactici, etc. The application of distiller yeasts producing antibacterial peptides enhances the ethanol yield in cases of bacterial contamination. Recombinant yeasts producing the antibacterial peptides pediocin and plantaricin can successfully substitute the available industrial yeast strains upon ethanol production.

Conformational Differences between Active Angiotensins and Their Inactive Precursors.

The peptide conformation in the context of a protein polypeptide chain is influenced by proximal amino acid residues. However, the mechanisms of this interference remain poorly understood. We studied the conformation of angiotensins 1, 2 and 3, which are produced naturally in a sequential fashion from a precursor protein angiotensinogen and contain an identical peptide core structure. Using the example of angiotensins 1, 2 and 3, it was shown that similar amino acid sequences may have significant conformational differences in various molecules. In order to assess the conformational changes, we developed a panel of high-affinity mouse monoclonal antibodies against angiotensins 1, 2 and 3 and studied their cross-reactivity in indirect and competitive ELISAs. It was found that the conformations of inactive angiotensin1 and the corresponding fragment of angiotensinogen are similar; the same is true for the conformations of active angiotensins 2 and 3, whereas the conformations of homologous fragments in the active and inactive angiotensins differ significantly.

[Effects of recombinant mechano-dependent growth factor against the background of chronic alcoholisation in rats].

In alcoholised rats, proliferation of satellite cells consistently decreased as well as the number of myonuclei, while phosphorylation of p90RSK became reduced. The mechano-growth factor abministration increased the proliferate activity of the myogenic precursors and restored the myonuclei pool. Phosphorylation of p90RSK increased too.

[Extremophilic microorganisms: biochemical adaptation and biotechnological application (review)].

The analysis of modern data on biochemical adaptation of microorganisms for living in extreme conditions is presented in this review. Special attention has been paid to the analysis of adaptive responses of microorganisms to the conditions of increased radiation at the molecular and cellular levels. The data on the practical application of extremophils and extremoenzymes, synthesized by them, biologically active substances, biopolymers and so on, have been systematized.

[Selection of DNA aptamers, specifically interacting with fibrillar form of the yeast Sup35 protein].

Single-stranded DNA aptamers interacting with fibers formed by the Sup35 protein of Saccharomyces cerevisiae were obtained by the SELEX procedure. Specificity of interaction with Sup35p is investigated for 10 from total of 40 selected aptamers. It is shown that 9 aptamers bind to fibrillar Sup35p and not to monomeric form of the protein. The rate of dissociation constant of aptamer-fiber complex varies from 0.1 to 1 microM for different aptamers. Selected aptamers can be used to study prionization of Sup35.

Nitrate reductases: structure, functions, and effect of stress factors.

Structural and functional peculiarities of four types of nitrate reductases are considered: assimilatory nitrate reductase of eukaryotes, as well as cytoplasmic assimilatory, membrane-bound respiratory, and periplasmic dissimilatory bacterial nitrate reductases. Arguments are presented showing that eukaryotic organisms are capable of nitrate dissimilation. Data concerning new classes of extremophil nitrate reductases, whose active center does not contain molybdocofactor, are summarized.

[Dissimilatory nitrate reduction in fungi under conditions of hypoxia and anoxia: a review].

Recent progress in studies of anaerobic nitrate reduction and nitrous oxide formation in fungi has been reviewed. Current understanding of the biochemistry of nitrate and nitrite reduction to nitrous oxide and ammonium under oxygen limitation is presented, with emphasis on patterns of fungal co-denitrification, properties of the enzymes involved, and prevalence of nitrate respiration among fungal species.

Characterization of molybdenum-free nitrate reductase from haloalkalophilic bacterium Halomonas sp. strain AGJ 1-3.

Nitrate reductase from the haloalkalophilic denitrifying bacterium Halomonas sp. strain AGJ 1-3 was isolated and purified to homogeneity. The isolated enzyme belongs to a novel family of molybdenum-free nitrate reductases. It presents as a 130-140 kD monomeric protein with specific activity of 250 micromol/min per mg protein. The enzyme reduces not only nitrate, but also other anions, thus showing polyoxoanion reductase activity. Enzyme activity was maximal at pH 7.0 and 70-80 degrees C.

Isolation, purification, and characterization of nitrate reductase from a salt-tolerant Rhodotorula glutinis yeast strain grown in the presence of tungsten.

The salt-tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35-45 degrees C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.

[Properties of nitrate reductase from Fusarium oxysporum 11dn1 fungi grown under aerobic and anaerobic conditions].

Production of nitrate reductase was studied in 15 species of microscopic fungi grown on a nitrate-containing medium. Experiments were performed with Fusarium oxysporum 11dn1, a fungus capable of producing nitrous oxide as the end product of denitrification. Moreover, a shift from aerobic to anaerobic conditions of growth was accompanied by a sharp increase in the activity of nitrate reductase. Studies of nitrate reductase from the mycelium of Fusarium oxysporum 11dn1, grown under aerobic and anaerobic conditions, showed that this enzyme belongs to molybdenum-containing nitrate reductases. The enzymes under study differed in the molecular weight, temperature optimum, and other properties. Nitrate reductase from the mycelium grown under aerobic conditions was shown to belong to the class of assimilatory enzymes. However, nitrate reductase from the mycelium grown anaerobically had a dissimilatory function. An increase in the activity of dissimilatory nitrate reductase, observed under anaerobic conditions, was associated with de novo synthesis of the enzyme.