RESUMO
The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for super-transformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-gamma and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L(-1) range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFalpha1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.
Assuntos
Calnexina/genética , Calnexina/metabolismo , Dosagem de Genes , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , 6-Fitase/genética , 6-Fitase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Calnexina/biossíntese , Calnexina/química , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Clonagem Molecular , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Interferon gama/genética , Interferon gama/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Albumina Sérica/genética , Albumina Sérica/metabolismo , Transformação GenéticaRESUMO
gamma-Interferon (IFNgamma) is poorly secreted from recombinant Hansenula polymorpha cells in the form of hyperglycosylated molecules. A selection of four secretory-pathway genes was assessed for possible impact on secretion improvements. For assessment, rDNA integration vectors harbouring the H. polymorpha-derived KAR2, PDI, SSO2 or CNE1 gene were co-integrated along with the IFNgamma-integration/expression vector. Overexpression of CNE1 resulted in an increased secretion of the cytokine, predominantly consisting of molecules of distinct size. Deglycosylation with PGNaseF resulted in an M(r) reduction of the secreted IFNgamma corresponding to the removal of two N-linked glycoside chains. Coexpression of KAR2, PDI or SSO2 exhibited no effect.
RESUMO
An Arxula adeninivorans integration vector was applied to a range of alternative yeast species including Saccharomyces cerevisiae, Debaryomyces hansenii, Debaryomyces polymorphus, Hansenula polymorpha and Pichia pastoris. The vector harbours a conserved A. adeninivorans-derived 25S rDNA sequence for targeting, the A. adeninivorans-derived TEF1 promoter for expression control of the reporter sequence, and the Escherichia coli-derived hph gene conferring resistance against hygromycin B for selection of recombinants. Heterologous gene expression was assessed using a green fluorescent protein (GFP) reporter gene. The plasmid was found to be integrated into the genome of the various hosts tested; recombinant strains of all species exhibited heterologous gene expressions of a similar high level.
Assuntos
Regulação da Expressão Gênica , Vetores Genéticos , Saccharomycetales/genética , Leveduras/genética , Clonagem Molecular , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Transformação Genética , Leveduras/metabolismoRESUMO
A method that has been successfully used to generate recombinant Hansenula polymorpha strains by transformation with rDNA-targeting vectors was applied in the present study to a range of alternative yeast hosts, using vectors with an H. polymorpha-derived integration sequence. The dimorphic yeast Arxula adeninivorans, which is currently being assessed for heterologous gene expression, was the main focus of the study. As in H. polymorpha, it was possible to co-integrate more than a single plasmid carrying an expressible gene. Additionally, the vectors were examined in two further species, Pichia stipitis and Saccharomyces cerevisiae. Based on these results the design of a 'universal' fungal vector appears to be feasible.
Assuntos
DNA Ribossômico/genética , Vetores Genéticos , Pichia/genética , Saccharomycetales/genética , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Fúngico , DNA Recombinante/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Genes Reporter , Teste de Complementação Genética , Genoma Fúngico , Fases de Leitura Aberta , Pichia/metabolismo , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido Nucleico , Transformação GenéticaRESUMO
The mannan-binding lectin (MBL) is a serum protein, which is involved in the immune defence against viruses, bacteria and parasites. Children who have mutations in the MBL gene that lead to a MBL deficiency are more susceptible to infectious diseases and are more likely to suffer from severe malaria. In this report we investigate the interaction between MBL and the proteins of red blood cells infected with the parasite Plasmodium falciparum. Protein extracts were separated on MBL-sepharose columns. After the elution of bound material, the proteins were detected either by Western blot with human antibodies, or radioactive labelling with 35S-methionine or 3H-glucosamine. MBL recognises proteins of P. falciparum-infected erythrocytes that are immunogenic in humans, parasite-derived and glycosylated. Whether the proteins identified in the different assays are identical remains to be explored. MBL added to in vitro cultures of P. falciparum, however, does not inhibit parasite growth. The positive effect of MBL in the blood of malaria patients could be caused by detoxification of parasite products.
