[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.

[Multigene families of endo-(1-4)-beta-xylanases of Penicillium canescens].

Four novel genes of the enzymes of the endoxylanase (EC 3.2.1.8) families found in the mycelial fungus Penicillium canescens have been cloned. The xylB, xylC, and xylD genes encode endoxylanases of glycosyl hydrolase family 11; the xylEgene, those of family 10. In the promoter region of the xylB, xylC, and xylD genes, the binding sequences for the protein activator of xylanolytic gene transcription have been found; the promoter region of the xylB gene contains the binding sequences for the catabolite repression protein. Since the TATAA sequence, which is an element of the minimal eukaryotic promoter, has not been found in the promoter region of the xylC gene, in contrast to those of the xylB and xylD genes, it may be assumed that this gene is silent. Comparative phylogenetic analysis has shown that the cloned genes are highly homologous to some endoxylanase genes of mycelial fungi of the genera Penicillium and Aspergillus. However, within the species P. canescens, they exhibit a low homology both within and between families, and they diverge into different branches of the phylogenetic tree, which suggest divergence of the genes of this group at an early stage of evolution.

[The mutational analysis of carbon catabolite repression in filamentous fungus Penicillium canescens].

Penicillium canescens strain F178 is a natural producer of beta-galactosidase and endo-1,4-beta-xylanase. The transcription of genes bgaS and xylA, coding for these proteins, is subject to carbon catabolite repression. The system for selective isolation of regulatory mutants in P. canescens is developed. Two strains from the mutant collection are studied in details. It is shown that both mutations can be complement by creA gene of P. canescens, encoding global regulator of carbon catabolite repression in filamentous fungi. creA(-) alleles contain frameshift mutations in C-domain of CreA. Gene xylA is derepressed in mutants at transcription level in the presence of D-glucose. A transcription of creA gene in mutants is also derepressed proving effect of autoregulation for this gene.

[Transcriptional regulator of carbon catabolite repression CreA in filamentous fungus].

Penicillium canescens strain F178 is a natural producer of beta-galactosidase and endo-1,4-beta-xylanase. Tanscription of genes bgaS and xylA coding for these proteins is subject to carbon catabolite repression which proceed mainly in filamentous fungi by transcriptional repressor CreA. creA gene of P. canescens was cloned. It was demonstrated that creA transcription is also subject to carbon catabolite repression. CreA protein remains intranuclear independently of nature of carbon source and glucose concentration in culture medium. In vitro experiments confirm availability of four CreA-binding sites in bgaS promoter, four sites in xylA promoter and one such site in creA promoter.

[A heterologous production of the Trametes hirsuta laccase in the fungus Penicillium canescens].

A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta laccase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches 3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency ofheterologous secretion. The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072.

[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.

Enzymological properties of endo-(1-4)-beta-glucanase Eg12p of Penicillium canescens and characteristics of structural gene egl2.

Gene egl2 of secreted endo-(1-4)-beta-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding beta-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60 degrees C, respectively, exhibited specific activity of 33 IU, and had K(m) and V(max) in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 micromol/sec per mg, respectively.

[Cloning of the Penicillium canescens endo-1,4-beta-glucanase gene egl3 and the characterization of the recombinant enzyme].

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-beta-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for beta-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54 degrees C, respectively. The Km and Vm values for CMC hydrolysis were determined; they amounted to 17.1 g/1 and 0.31 microM/(mg s), respectively.

[Coregulation of alternative transcripts of the STA2 gene in Saccharomyces cerevisiae]. / Koreguliatsiia al'ternativnykh transkriptov gena STA2 drozhzhei Saccharomyces cerevisiae.

It is known that upon STA2 gene expression in Saccharomyces cerevisiae cells, two transcripts of different lengths are formed. These fragments contain different start codons of translation (AUG1 and AUG2) located in the same reading frame. The ratio between expression levels of proteins translated from AUG1 and AUG2 was invariably about 2:7 and did not depend on the choice of the reporter product (secreted glucoamylase (GA) or beta-galactosidase accumulated in cells). Neither this ratio depended on glucose repression/derepression. Based on the assumed proportional relationship between levels of transcription and translation of the STA2 gene in yeast cells, all these results cumulatively indicate that the two STA2 transcripts are coregulated. The production of secreted GA was also shown to be markedly stimulated at the posttranscriptional level under conditions of glucose repression.

Immune response modulation by recombinant peptides expressed in virus-like particles.

Aspergillus fumigatus, a ubiquitous fungus, is implicated in the pathogenesis of a number of clinically different allergic diseases in man, including allergic bronchopulmonary aspergillosis. Peptide-based immunotherapy may offer an alternative treatment strategy for the management of allergic disease. The objective of this study was to alter the allergen-specific immune response using dominant T cell epitopes of a major A. fumigatus allergen, Asp f2, expressed in yeast as virus-like particles (VLP). The T cell epitopes of Asp f2, recognized in mice with an H-2d background, were determined by producing T-cell hybridomas. Two dominant T cell epitopes, aa60--71 and aa235--249, were identified and expressed in a yeast VLP system. To induce tolerance VLP-peptides were injected subcutaneously into mice previously immunized with recombinant Asp f2. The T cell immune response was abrogated totally in 3 weeks following a single injection of VLP but was restored 2 months later following intranasal antigen exposure. T-cell depletion resulted in the reduction of 20-30% of all antigen-specific immunoglobulin classes. Thus, recombinant peptides expressed in the VLP system can be used successfully in the modulation of Asp f2-induced immune response in mice, although a single administration is not sufficient to maintain a state of tolerance for a long period of time.

[Cloning the Saccharomycopsis fibuligera alpha-amylase gene and its expression in Saccharomyces cerevisiae]. / Klonirovanie gena alpha-amilazy drozhzhei Saccharomycopsis fibuligera i ego ékspressiia v Saccharomyces cerevisiae.

The alpha-amylase gene of yeast Saccharomycopsis fibuligera was cloned, partially sequenced, and expressed in Saccharomyces cerevisiae. Four amino acid substitutions were identified in the enzyme structure as compared to the earlier cloned gene. The Saccharomycopsis fibuligera alpha-amylase gene was expressed in Saccharomyces cerevisiae under the control of its own promoter. The native alpha-amylase signal peptide provided an efficient secretion of the enzyme by Saccharomyces cerevisiae. The efficiency of the alpha-amylase secretion was 98%. The pH optimum of the enzyme secreted by Saccharomyces cerevisiae was 4.0.

[Mutational analysis of the starch utilization system in the yeast Saccharomyces cerevisiae]. / Mutatsionnyi analiz sistemy utilizatsii krakhmala u drozhzhei Saccharomyces cerevisiae.

Seven mutants of Saccharomyces cerevisiae deficient in production of extracellular glucoamylase have been analyzed. For each of the seven a monogenic pattern of inheriting the mutant phenotype has been observed. The mutations have been shown to map within five different genetic loci, three independent mutations affecting the STA2 locus and the other four residing in four formerly unidentified genes. As expected, the sta2 mutants recover the wild phenotype when transformed with a STA2-bearing multicopy plasmid. Such reversion has also been observed for the transformed stall mutant. Unlike the others, the sta16 mutant is unable to secrete heterologous alpha-amylase encoded by a plasmid-borne DNA fragment. All the mutants have a moderately reduced ability to secrete the invertase and acid phosphatase.

["Illegal" transformation of red adenine-dependent mutants of the yeast Pichia pinus by the pYE(ADE2)2 plasmid]. / "Nezakonnaia" transformatsiia krasnykh adeninzavisimykh mutantov drozhzhei Pichia pinus plazmidoi pYE(ADE2)2.

The red adenine-dependent mutants ade1 of the yeast Pichia pinus blocked in the VI step of adenine biosynthesis (lack of AIR-carboxylase) and ade2 mutants blocked in the VII step of adenine biosynthesis (lack of SAIKAR-synthase) were transformed with the plasmid pYE(ADE2)2 containing ADE2 gene of Saccharomyces cerevisiae encoding AIR-carboxylase. The appearance of white Ade+ clones with the frequency 2-7.10(-8) (which is ten-fold higher than reversion frequency) was only observed in the case of ade2 transformation. Genetic analysis points to connection of the "illegitimate" transformants' appearance with the change in the mutant ade2 locus or in a locus closely linked to the former. Ade+ phenotype was stable during 20 generations of mitotic budding. Southern blotting assay of transformant chromosomal DNA indicates that reconstitution of ade2 defective gene is related with its "correction", owing to integration of pYE(ADE2)2 sequence in the vicinity of the mutant locus.

[A new vector for cloning of genes and replicons in yeasts]. / Novyi vektor dlia klonirovaniia genov i replikonov v drozhzhakh.

A novel Escherichia coli-Saccharomyces cerevisiae shuttle vector lambda MAN78 has been constructed. The vector contains phage lambda 47.1 DNA, Sacch. cerevisiae chromosomal segment with TRP1 gene and the yeast ARS1 replicator. This vector may be propagated as a phage and, similar to parental lambda 47.1, allows direct selection of large DNA inserts (15-24 kbp) in E. coli. lambda MAN78 can efficiently transform LiCl-treated yeast cells (3-5 X 10(3) transformants per 1 microgram DNA). Replication of hybrid molecules in E. coli cells does not influence the ability of the molecules to transform yeast cells and replicate in the cells.