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
We have cloned and characterized a 2,778-kb XbaI segment of the mitochondrial genome of the locust, Locusta migratoria. It harbours portions of the ND4 and the ND1 genes, the entire genes for ND6, ND4L and cytochrome b, and the genes for three mitochondrial tRNAs. The genes are arranged in an order which is conserved between orthopteran and dipteran insects. The analysis of the cytochrome b sequence, and its comparison with other systems, supports the current model structure for this polypeptide.
Assuntos
Grupo dos Citocromos b/genética , DNA Mitocondrial/genética , Genes de Insetos , Gafanhotos/genética , NADH Desidrogenase/genética , RNA de Transferência de Serina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Grupo dos Citocromos b/química , Insetos/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA de Transferência de Prolina/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
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
The lipid-transport lipoprotein, lipophorin (Lp), an abundant plasma protein of insects, is synthesized in the fat body of Locusta migratoria as a polypeptide of relative mass (Mr) = 85 000. No posttranslational modification could be detected. Precipitation of this protein leads to formation of a product of Mr about 240 000, which fails to dissociate under treatment with sodium dodecyl sulfate and beta-mercaptoethanol. In adult female locusts, synthesis and secretion of protein by the fat body increases three- to four-fold from emergence until day 7 of adult life, Lp representing 15-30% of the total. In the subsequent vitellogenic stage (days 7-14 or later), a further impressive increase in protein output up to five times the previtellogenic rate is observed, followed by a decrease to the previtellogenic level at the end of the cycle. This fluctuation is mainly due to production of the yolk precursor protein, vitellogenin (Vg), which makes up some 60% of the secreted protein at the peak of the cycle. Production of Lp does not follow these dramatic changes. Inactivation of the corpora allata, the source of juvenile hormone (JH), by treatment with precocene, prevents the synthesis of Vg in females, and diminishes the production of other proteins, including Lp, in both sexes. Normal secretion patterns can be restored by application of the JH analog, methoprene. It is concluded that, whereas the synthesis of Vg is dependent upon and intensely stimulated by JH, the synthesis of Lp is stimulated only to a minor extent, in common with other proteins.