RESUMO
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.
Assuntos
Hirudinas/metabolismo , Pichia/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes , Braquiúros/genética , Fermentação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Glucana 1,4-alfa-Glucosidase/biossíntese , Glucana 1,4-alfa-Glucosidase/genética , Hirudinas/biossíntese , Hirudinas/genética , Hormônios de Invertebrado/biossíntese , Hormônios de Invertebrado/genética , Fator de Acasalamento , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Biossíntese Peptídica , Peptídeos/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Citotoxicidade Imunológica , Antígenos H-2/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Adjuvantes Imunológicos , Animais , Antígenos CD8/análise , Relação Dose-Resposta Imunológica , Feminino , Antígenos de Superfície da Hepatite B/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peptídeos/imunologia , Solubilidade , Vacinas SintéticasRESUMO
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.
Assuntos
Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Leveduras/genética , Leveduras/metabolismo , Expressão Gênica , Vetores Genéticos , Kluyveromyces/genética , Kluyveromyces/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
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.