RESUMEN

As a product of western world biotechnology the yeast (Saccharomyces cerevisiae) hepatitis B vaccine was introduced as antigenic subtype adw2. However, an HBsAg/adw2-vaccine may provide a good but not "optimal" immunologic response for infection with heterologous virus strains. The availability of the yeast Hansenula polymorpha HBsAg in three different antigenic forms (adw2, ayw3 and adr) enabled us to investigate the influence of variant amino acids in the binding of immune anti-HBs after vaccination. Hansenula-derived HBsAg was standardised on the basis of protein content at >95% purity. Standardisation was controlled by monoclonal anti-HBs binding in a well-conserved region. Sera were obtained after immunisation with type adw, ayw and adr vaccines. Direct binding of immune antibodies to homologous antigen (in EIA) was higher than to heterologous antigen except for the adr-related antibodies. Since the binding of the WHO reference anti-HBs was strongly reduced for the ayw and adr compared to the adw antigen, a similar binding profile for the three antigens on protein basis could result in 2-3-fold different anti-HBs level expressed in IU/l. Inhibition of Hansenula-derived HBsAg binding to solid phase monoclonal anti-HBs in enzyme immunoassays after incubation with serum anti-HBs confirmed the differential binding of serum anti-HBs with variant Hansenula-derived HBsAg. This variant (antigenic subtype) dependent reactivity of anti-HBs in immunoassays in combination with a variant specific WHO standard may limit the application of the threshold levels of 10 and 100 IU/l for seroconversion and seroprotection.

Asunto(s)

Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Vacunas Sintéticas/inmunología , Adulto , Vectores Genéticos , Anticuerpos contra la Hepatitis B/inmunología , Humanos , Pichia , Proteínas Recombinantes de Fusión/inmunología , Vacunación

RESUMEN

The methylotrophic yeast Hansenula polymorpha has been developed as an efficient production system for heterologous proteins. The system offers the possibility to cointegrate heterologous genes in anticipated fixed copy numbers into the chromosome. As a consequence co-production of different proteins in stoichiometric ratios can be envisaged. This provides options to design this yeast as an industrial biocatalyst in procedures where several enzymes are required for the efficient conversion of a given inexpensive compound into a valuable product. To this end recombinant strains have been engineered with multiple copies of expression cassettes containing the glycolate oxidase (GO) gene from spinach and the catalase T (CTT1) gene from S. cerevisiae. The newly created strains produce high levels of the peroxisomal glycolate oxidase and the cytosolic catalase T. The strains efficiently convert glycolate into glyoxylic acid, oxidizing the added substrate and decomposing the peroxide formed during this reaction into water and oxygen.

Asunto(s)

Oxidorreductasas de Alcohol/genética , Catalasa/genética , Expresión Génica , Pichia/genética , Proteínas Recombinantes/biosíntesis , Oxidorreductasas de Alcohol/metabolismo , Catalasa/metabolismo , Catálisis , Citosol/enzimología , Fermentación , Glicolatos/metabolismo , Glioxilatos/metabolismo , Microbiología Industrial , Microcuerpos/enzimología , Pichia/enzimología , Saccharomyces cerevisiae/enzimología , Spinacia oleracea/enzimología

RESUMEN

A DNA sequence coding for a subtype of the hirudin variant HV1 was expressed in the methylotrophic yeast Hansenula polymorpha from a strongly inducible promoter element derived from a gene of the methanol metabolism pathway. For secretion, the coding sequence was fused to the KEX2 recognition site of three different prepro segments engineered from the MF alpha 1 gene of Saccharomyces cerevisiae, the glucoamylase (GAM1) gene of Schwanniomyces occidentalis and the gene for a crustacean hyperglycemic hormone from the shore crab Carcinus maenas. In all three cases, correct processing of the precursor molecule and efficient secretion of the mature protein were observed. In fermentations on a 10-1 scale of a transformant strain harbouring a MF alpha 1/hirudin-gene fusion yields in the range of grams per litre could be obtained. The majority of the secreted product was identified as the full-length 65-amino-acid hirudin. Only small amounts of a truncated 63-amino- acid product, frequently observed in S. cerevisiae-based expression systems, could be detected.

Asunto(s)

Hirudinas/metabolismo , Pichia/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos , Braquiuros/genética , Fermentación , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Glucano 1,4-alfa-Glucosidasa/genética , Hirudinas/biosíntesis , Hirudinas/genética , Hormonas de Invertebrados/biosíntesis , Hormonas de Invertebrados/genética , Factor de Apareamiento , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Biosíntesis de Péptidos , Péptidos/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética

RESUMEN

Plasmids without an origin of replication, but bearing the URA3 gene of Saccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously in Hansenula polymorpha, indicating that parts of the S. cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function in H. polymorpha. Such plasmids, replicated in low copy number in monomeric conformation, could be rescued in E. coli, and showed a low mitotic stability under selective and non-selective conditions. Selective propagation of such transformants, however, led to the integration of plasmid sequences into the H. polymorpha genome. The integration event usually occurred in high copy number (approx. 30-50) at a single non-homologous site of the genome. The plasmid sequences were found to be present in tandem array and stable under non-selective conditions. In contrast, the use of homologous URA3 gene under similar conditions led to low-copy-number transformants.

Asunto(s)

Vectores Genéticos/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Pichia/genética , Plásmidos/genética , Transformación Genética/genética , Replicación del ADN/genética , ADN de Hongos/análisis , Amplificación de Genes , Genes Fúngicos/genética , Marcadores Genéticos , Saccharomyces cerevisiae/genética

Asunto(s)

Metanol , Pichia/genética , Secuencia de Bases , Clonación Molecular , ADN Recombinante , Fermentación , Vectores Genéticos , Metanol/metabolismo , Datos de Secuencia Molecular , Pichia/metabolismo , Proteínas Recombinantes/genética

RESUMEN

Immunization with soluble proteins only rarely induces a specific response of CD8+ CTL. We describe experiments that demonstrate the efficient and specific in vivo priming of CTL in BALB/c mice immunized with soluble hepatitis B virus (HBV)-derived surface (S) protein. A single (s.c., i.p. or i.v.) injection of a low dose (30 ng to 3 micrograms per mouse) of recombinant S protein particles without adjuvants induced a CTL response. This specific cytotoxic response was read out against a panel of different S protein-expressing transfected mouse cell lines. Effector cells of this response were Ld-restricted, CD3+ CD4- CD8+ CTL. H-2d/Ld+ (BALB/c, C.B-17) mice were responders; H-2d/Ld- (dm2) mutant mice and H-2b (C57BL/6) mice were nonresponders. Injections of various dosages of a S protein-derived, immunogenic, synthetic peptide into BALB/c mice by various routes did not prime CTL. After incorporation of S protein particles into IFA or aluminum hydroxide, these protein Ag lost their ability to specifically stimulate CTL in vivo. After priming of mice with S protein emulsified in IFA or adsorbed to aluminum hydroxide boost injections with native S protein particles were inefficient in stimulating a specific CTL response. These findings are of relevance for the design of synthetic subunit vaccines for which specific stimulation of CD8+ T effector functions is desired.

Asunto(s)

Citotoxicidad Inmunológica , Antígenos H-2/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Adyuvantes Inmunológicos , Animales , Antígenos CD8/análisis , Relación Dosis-Respuesta Inmunológica , Femenino , Antígenos de Superficie de la Hepatitis B/química , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Péptidos/inmunología , Solubilidad , Vacunas Sintéticas

RESUMEN

The methylotrophic yeasts have been the subject of intensive studies, because of their highly regulated methanol metabolism and the biogenesis of peroxisomes. We investigated the 5' regulatory region of the MOX gene from the yeast, Hansenula polymorpha, encoding the peroxisomal methanol oxidase, the key enzyme of methanol metabolism. This tightly regulated yeast promoter of approximately 1.5 kb is unusually large, and also of remarkable strength under inducing conditions, belonging to the strongest yeast promoters yet described. Deletion analyses revealed a complex promoter structure composed of several sequence elements with positive and negative regulatory effects on reporter gene expression and a pronounced cooperation between the elements. Specific binding of several factors was detected in vitro by gel retardation and DNase I footprinting experiments. On the basis of deletion data, two binding sites could be identified as upstream activation sequences (UAS1 and UAS2) and one binding site as an upstream repressing sequence (URS1).

Asunto(s)

Oxidorreductasas de Alcohol/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Pichia/enzimología , Pichia/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Oxidorreductasas de Alcohol/biosíntesis , Secuencia de Bases , ADN de Hongos/genética , ADN de Hongos/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Mapeo Restrictivo , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Transcripción Genética

RESUMEN

The ura3 gene of Hansenula polymorpha was cloned, sequenced and used to generate a ura3 mutant from the wild-type strain of this yeast via integrative mutagenesis. The Tn5 neomycin-resistance marker (neo) under control of the ADH1 promoter from Saccharomyces cerevisiae served as a transformation marker. The results show that gene replacement can be achieved in H. polymorpha, a yeast with a high level of non-homologous integration.

Asunto(s)

Genes Fúngicos/genética , Marcadores Genéticos , Orotidina-5'-Fosfato Descarboxilasa/genética , Pichia/genética , Transformación Genética/genética , Clonación Molecular , Eliminación de Gen , Mutagénesis Sitio-Dirigida , Pichia/enzimología , Análisis de Secuencia de ADN

Asunto(s)

Pichia/genética , Biotecnología , Mapeo Cromosómico , ADN Recombinante/genética , Expresión Génica , Vectores Genéticos , Pichia/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Especificidad de la Especie

RESUMEN

The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeast Saccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application. In the following review a selection from the different yeast systems is described and compared.

Asunto(s)

Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Levaduras/genética , Levaduras/metabolismo , Expresión Génica , Vectores Genéticos , Kluyveromyces/genética , Kluyveromyces/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

The methylotrophic yeast Hansenula polymorpha belongs to a limited number of non-Saccharomyces yeast species used as hosts for heterologous gene expression. It has successfully been applied for the production of hormones, antigens and enzymes. The system excells by mitotically stable recombinant strains, high productivity and faithful processing of the produced polypeptides. The favourable characteristics of this microorganism for protein production at an industrial scale are described in the following article focusing on some recent representative examples.

RESUMEN

An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co-express both the L (preS1-S2-S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol-inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5-8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle-specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H polymorpha-derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Antígenos de Superficie de la Hepatitis B/biosíntesis , Pichia/genética , Secuencia de Bases , Western Blotting , Medios de Cultivo , ADN de Hongos/química , Fermentación , Vectores Genéticos , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Immunoblotting , Datos de Secuencia Molecular , Pichia/metabolismo , Plásmidos , Regiones Promotoras Genéticas , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Mapeo Restrictivo

RESUMEN

We have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein. Transformants of H. polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified. Yields of up to 1.4g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter.

Asunto(s)

Clonación Molecular/métodos , Glucano 1,4-alfa-Glucosidasa/genética , Pichia/genética , Saccharomycetales/enzimología , Secuencia de Aminoácidos , Fermentación , Genes Fúngicos , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Glucano 1,4-alfa-Glucosidasa/metabolismo , Datos de Secuencia Molecular , Pichia/enzimología , Plásmidos

RESUMEN

The DAS gene of Hansenula polymorpha was expressed in Saccharomyces cerevisiae under the control of different promoters. The heterologously synthesized dihydroxyacetone synthase (DHAS), a peroxisomal enzyme in H. polymorpha, shows enzymatic activity in baker's yeast. The enzyme was imported into the peroxisomes of S. cerevisiae not only under the appropriate physiological conditions for peroxisome proliferation (oleic acid media), but also in glucose-grown cells where it induced the enlargement of the few peroxisomes present. This growth process was not accompanied by an increase in the number of microbodies, which suggests a separate control mechanism for peroxisomal proliferation.

Asunto(s)

Transferasas de Aldehído-Cetona , Regulación Fúngica de la Expresión Génica , Microcuerpos/enzimología , Pichia/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Transferasas/biosíntesis , Oxidorreductasas de Alcohol/biosíntesis , Oxidorreductasas de Alcohol/genética , Western Blotting , Fraccionamiento Celular , Inmunohistoquímica , Microcuerpos/metabolismo , Microcuerpos/ultraestructura , Microscopía Electrónica , Pichia/enzimología , Pichia/ultraestructura , Plásmidos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/ultraestructura , Transcripción Genética , Transferasas/análisis , Transferasas/genética , Transformación Genética

Asunto(s)

Genes Fúngicos , Metanol/metabolismo , Saccharomycetales/genética , Regulación de la Expresión Génica , Saccharomycetales/metabolismo

RESUMEN

The structural gene and the regulatory DNA sequence of the yeast Hansenula polymorpha methanol oxidase have been isolated. According to the nucleotide sequence data obtained, the structural gene encodes a 664 amino acids long protein, contains no intervening sequences, and the 5'- and 3'-non-coding region contains several sequences implicated in transcription initiation and termination in the yeast Saccharomyces cerevisiae. Although the methanol oxidase is translocated to the peroxisomes, no cleavable signal sequence was found at the N-terminus of the protein.

Asunto(s)

Oxidorreductasas de Alcohol/genética , Ascomicetos/genética , Clonación Molecular , Pichia/genética , Secuencia de Aminoácidos , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , Endonucleasas/metabolismo , Conformación de Ácido Nucleico , Pichia/enzimología , Poli A/análisis , Biosíntesis de Proteínas , ARN/análisis , ARN Mensajero , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Transcripción Genética

RESUMEN

A gene library from the methanol utilizing yeast Hansenula polymorpha, constructed in a lambda Charon4A vector, was used to clone the gene encoding a key methanol assimilating enzyme, dihydroxyacetone synthase (DHAS) by differential plaque hybridization. The nucleotide sequence of the 2106 bp structural gene and the 5' and 3' non-coding regions was determined. The deduced amino acid sequence of the protein is in agreement with the apparent molecular weight and amino acid composition of the purified protein. The codon bias is not so pronounced as in some Saccharomyces cerevisiae genes.

Asunto(s)

Transferasas de Aldehído-Cetona , Ascomicetos/genética , Clonación Molecular , Metanol/metabolismo , Pichia/genética , Transferasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , Endonucleasas/metabolismo , Genes , Hibridación de Ácido Nucleico , Pichia/enzimología , Biosíntesis de Proteínas , ARN Mensajero/análisis , Saccharomyces cerevisiae/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Transcripción Genética

RESUMEN

The biosynthesis of methanol oxidase, a peroxisomal enzyme in the methanol-utilizing yeast Hansenula polymorpha, was studied in vitro. Translation of Hansenula mRNA in a rabbit reticulocyte lysate yields methanol oxidase protein in high amounts. The apparent molecular mass of the protein was found to be identical to the subunit of the functional multimeric enzyme, which indicates the absence of an N-terminal extension typical of most transported proteins. The regulation of methanol oxidase by glucose repression and depression as well as by induction of methanol was shown to be controlled at the level of transcription. Two mutants of Hansenula polymorpha, unable to grow on methanol as a carbon and energy source were shown to be affected in methanol oxidase synthesis.

Asunto(s)

Oxidorreductasas de Alcohol/genética , Ascomicetos/genética , Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Pichia/genética , Oxidorreductasas de Alcohol/biosíntesis , Proteínas Fúngicas/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Metanol/farmacología , Microcuerpos/enzimología , Pichia/enzimología , Biosíntesis de Proteínas , ARN de Hongos/genética , ARN Mensajero/genética

RESUMEN

Saccharomyces cerevisiae was transformed with the Escherichia coli ompA gene coding for an outer membrane protein. Yeast transformants containing the pYTU101 plasmid, consisting of the ompA gene cloned in pSC101 and the HindIII-3 fragment of 2-microns DNA, express the foreign membrane protein. The protein synthesized in yeast has an Mr value very similar if not identical to that of the mature E. coli protein. The expressed protein is present in yeast mitochondrial and plasma membrane fractions. The yeast cell can tolerate about 250 molecules of the foreign membrane protein per cell, although the transformants show altered growth kinetics.

Asunto(s)

Proteínas Bacterianas/genética , Escherichia coli/genética , Proteínas de la Membrana/genética , Saccharomyces cerevisiae/genética , Compartimento Celular , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Genes , Genes Bacterianos , Mitocondrias/metabolismo , Peso Molecular , Plásmidos , Transformación Genética

RESUMEN

Transfer RNAs were isolated from plants representing mono- and dicotyledons: wheat embryos and lupin seeds. The two tRNA preparations were compared by polyacrylamide-gel-electrophoresis mapping. Transfer RNAs extracted from the separated spots were tested for acceptance of 11 amino acids. Comparison of electrophoretic mobility of respective tRNA species indicates the overall similarity of tRNA populations in the two plants studied. Especially, isoaccepting tRNAs for glycine, tyrosine and valine and some of isoacceptors of tRNAArg, tRNAAsp, tRNALeu, tRNALys and tRNAPhe occupy identical or closely similar positions on both polyacrylamide-gel maps. However, some tRNA isoacceptors from one population have no counterparts in the second one, which may indicate differences in their primary structures.

Asunto(s)

Plantas/análisis , ARN de Transferencia/análisis , Triticum/análisis , Electroforesis en Gel de Poliacrilamida