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
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.
RESUMO
We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.