RESUMEN
In order to show site-specific cleavage of fusion proteins with an engineered soluble yscF variant, we have constructed a fusion gene encoding eglinC from Hirudo medicinalis and the cellulose-binding domain from the Cellulomonas fimi exoglucanase (Cex). The two fusion partners were separated by a Lys-Arg-containing recognition sequence for the yeast endoprotease yscF (Kex2). The fusion protein (eglinC-Cex) was expressed intracellularly in Saccharomyces cerevisiae. After disruption of the cells eglin-C-Cex was shown to bind to cellulose when present in total crude cell lysates. This step efficiently removed the majority of contaminating host proteins. While immobilized on cellulose, eglin-C-Cex could be cleaved into the expected fragments. Upon cleavage the eglinC part was released from the cellulose, while the purification tag, i.e. the cellulose binding domain, stayed bound to the cellulose matrix.
Asunto(s)
Celulosa/metabolismo , Enzimas Inmovilizadas/metabolismo , Proproteína Convertasas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae , Serpinas , Subtilisinas , Secuencia de Aminoácidos , Secuencia de Bases , Celulosa/análogos & derivados , Clonación Molecular , Glucosidasas/genética , Glucosidasas/metabolismo , Datos de Secuencia Molecular , Plásmidos , Unión Proteica , Proteínas , Saccharomyces cerevisiae , Serina Endopeptidasas/genética , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/metabolismoRESUMEN
The prepro sequence of the yeast prepro-alpha-factor, usually referred to as the alpha-factor leader, has often been used for the efficient secretion of heterologous proteins from the yeast Saccharomyces cerevisiae. The alpha-factor leader consists of a 19-amino acid N-terminal pre or signal sequence followed by a 66-amino acid proregion. After removal of the signal sequence during membrane translocation, the proregion is cleaved from the precursor protein by the Kex2 endoprotease only in a late Golgi compartment. Here we report that a modified Kex2 enzyme, containing at the C-terminus the HDEL tetrapeptide, cleaves the proregion from the alpha-factor leader--human insulin like growth factor-1 fusion protein in the endoplasmic reticulum. The processing of pro-proteins earlier in the secretion pathway could be helpful in defining the cellular function of the proregions present naturally in various eucaryotic precursor proteins.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Proproteína Convertasas , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Serina Endopeptidasas/genética , Subtilisinas , Secuencia de Bases , Glicosilación , Datos de Secuencia Molecular , Mutación , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/metabolismo , Somatomedinas/metabolismoRESUMEN
The Saccharomyces cerevisiae KEX2 gene encodes the membrane-bound endoprotease yscF, which is responsible for the site-specific endoproteolytic cleavages at pairs of basic amino acid residues in the alpha-factor precursor. In order to obtain soluble yscF activity, a mutant KEX2 gene lacking 600 bp coding for the C-terminal 200 amino acids was constructed. Expression of the truncated KEX2 gene in yeast led to the secretion of an active soluble yscF protein (yscFs). The soluble yscF protein is able to efficiently cleave heterologous protein precursors in-vitro, as demonstrated for alpha-factor leader-hIGF1 and alpha-factor leader-hirudin fusion proteins.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proproteína Convertasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Serina Endopeptidasas/genética , Subtilisinas , Colorimetría , Fermentación , Hirudinas/metabolismo , Immunoblotting , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor de Apareamiento , Mutación , Péptidos/metabolismo , Plásmidos , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Serina Endopeptidasas/metabolismo , Solubilidad , Transformación GenéticaRESUMEN
The PDC1 gene coding for a pyruvate decarboxylase (PDC; EC 4.1.1.1) was deleted from the Saccharomyces cerevisiae genome. The resulting pdc1(0) mutants were able to grow on glucose and still contained 60 to 70% of the wild-type PDC activity. Two DNA fragments with sequences homologous to that of the PDC1 gene were cloned from the yeast genome. One of the cloned genes (PDC5) was expressed at high rates predominantly in pdc1(0) strains and probably encodes the remaining PDC activity in these strains. Expression from the PDC1 promoter in PDC1 wild-type and pdc1(0) strains was examined by the use of two reporter genes. Deletion of PDC1 led to increased expression of the two reporter genes regardless of whether the fusions were integrated into the genome or present on autonomously replicating plasmids. The results suggested that this effect was due to feedback regulation of the PDC1 promoter-driven expression in S. cerevisiae pdc1(0) strains. The yeast PDC1 gene was expressed in Escherichia coli, leading to an active PDC. This result shows that the PDC1-encoded subunit alone can form an active tetramer without yeast-specific processing steps.
Asunto(s)
Carboxiliasas/genética , Genes Fúngicos , Mutación , Piruvato Descarboxilasa/genética , Saccharomyces cerevisiae/genética , Northern Blotting , Southern Blotting , Clonación Molecular/métodos , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Escherichia coli/genética , Expresión Génica , Cinética , Plásmidos , Regiones Promotoras Genéticas , Piruvato Descarboxilasa/metabolismo , ARN de Hongos/genética , ARN de Hongos/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Ácido NucleicoRESUMEN
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.
Asunto(s)
Ascomicetos/enzimología , Transformación Genética , Levaduras/enzimología , alfa-Amilasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Expresión Génica , Datos de Secuencia Molecular , alfa-Amilasas/análisis , alfa-Amilasas/metabolismoRESUMEN
The PDC1 gene of Saccharomyces cerevisiae, encoding pyruvate decarboxylase was sequenced. The gene contains an open reading frame of 1647 base pairs. The codon usage shows the same strong bias as found for some other glycolytic enzymes. Transcription starts mainly at -30 and terminates 100 base pairs downstream of the termination codon. In some strains a second termination site, 46 base pairs upstream of the stop codon was observed. The function of the promoter region was analyzed by fusion to the bacterial structural gene encoding beta-lactamase (bla). On multicopy plasmid or integrated in the genome, the expression of the bla gene showed the regulation of the authentic PDC1 gene.