RESUMO
One of the major problems with the production of biotechnologically valuable proteins has been the purification of the product. For Escherichia coli and Saccharomyces cerevisiae, there are several techniques for the purification of intracellular proteins, but these are time consuming and often result in poor yields. Purification can be considerably facilitated, if the product is secreted from the host cell. In the work presented, we have constructed an expression vector (pSGNH2) for the secretion of protein disulfide isomerase (PDI; EC 5.3.4.1) from Aspergillus niger, in which the retention signal His-Asp-Glu-Leu (H-D-E-L) was modified to Ala-Leu-Glu-Gln (A-L-E-Q) via the polymerase chain reaction (PCR) method. The PDI gene was placed under the control of the A. oryzae alpha-amylase promoter. This expression vector was transformed into A. niger NRRL3, resulting in PDI secretion into the medium. The catalytic activity of overexpressed PDI from A. niger was indistinguishable from that of PDI isolated from bovine liver. With further strain improvement and optimization of culture conditions, it could be possible to raise the PDI production to the bioprocessing scale.
Assuntos
Aspergillus/genética , Isomerases de Dissulfetos de Proteínas/genética , Aspergillus/enzimologia , Aspergillus niger/metabolismo , Clonagem Molecular , Vetores Genéticos , Regiões Promotoras Genéticas , Isomerases de Dissulfetos de Proteínas/biossíntese , Proteínas Recombinantes/biossíntese , Transformação GenéticaRESUMO
The genomic pectin methylesterase (PME)-encoding gene (pmeA) from Aspergillus niger strain RH5344 was cloned by probing a genomic DNA library with a cDNA coding for PME. The recombinant phage clone was isolated and a 6-kb HindIII fragment was subcloned and characterized. The gene consists of seven exons and six introns. The nucleotide sequences of the coding regions were identical to those found in the pmeA cDNA. Cotransformation of A. niger was achieved with the vector, pAN7-1, and transformants were then tested for PME production. Transformants which produced more PME than the untransformed recipient strain were subjected to Southern-blot and Northern-blot analysis. The results show that there is a reasonable correlation between gene copy number, mRNA levels and PME production. PME was produced by A. niger transformants in an active 43-kDa form, which is similar to that of the mature protein isolated from the strain, RH5344. On the basis of the results of affinity labeling of PME with sugar-specific lectins and the amino acid sequence data, it has been revealed that PME is a glycoprotein and the protein-bound glycans are oligosaccharides with a high mannose content.
Assuntos
Aspergillus niger/genética , Hidrolases de Éster Carboxílico/genética , Genes Fúngicos , Marcadores de Afinidade , Sequência de Aminoácidos , Aspergillus niger/enzimologia , Sequência de Bases , Northern Blotting , Southern Blotting , Hidrolases de Éster Carboxílico/análise , Hidrolases de Éster Carboxílico/metabolismo , DNA Fúngico/genética , Expressão Gênica , Dados de Sequência Molecular , RNA Fúngico/genética , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico , Transformação GenéticaRESUMO
We have cloned a gene encoding a polygalacturonase (PG) in the filamentous fungus Aspergillus niger RH5344. The structural gene comprises 1141 bp coding for 362 amino acids and the open reading frame is disrupted by one intron of 52 bp. Eukaryotic consensus sequences for transcription regulation are found only in deviated forms. The biological functionality of the isolated PG gene was established by retransformation in A. niger and Aspergillus awamori. In addition, we have found that the PG protein of A. niger shares significant similarities with PG proteins from tomato and Erwinia carotovora. Comparison of the three enzymes revealed a highly conserved region in their C-terminal region probably comprising the elements of substrate binding and the catalytic centre.
Assuntos
Aspergillus niger/enzimologia , Poligalacturonase/genética , Sequência de Aminoácidos , Aspergillus niger/genética , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Fúngico/genética , Genes Fúngicos , Dados de Sequência Molecular , Pectobacterium carotovorum/genética , Plantas/genética , Poligalacturonase/química , Poligalacturonase/metabolismoRESUMO
A 1319 bp long cDNA encoding for a polygalacturonase (EC 3.2.1.15) from Aspergillus niger RH5344 comprises a single open reading frame of 1089 bp which includes the mature protein of 362 amino acids and an NH2-terminal signal peptide of 27 amino acids. The directly determined peptides of the mature polygalacturonase confirmed the sequence information deduced from the cDNA.
Assuntos
Aspergillus niger/genética , DNA Fúngico/análise , Glicosídeo Hidrolases/genética , Poligalacturonase/genética , Sequência de Aminoácidos , Aspergillus niger/enzimologia , Autoanálise , Sequência de Bases , Clonagem Molecular , Códon , Dados de Sequência Molecular , Poligalacturonase/biossínteseAssuntos
Proteínas Ribossômicas/isolamento & purificação , Cromatografia DEAE-Celulose/métodos , Cromatografia em Gel/métodos , Escherichia coli/metabolismo , Etilmaleimida , Cinética , Peso Molecular , Iniciação Traducional da Cadeia Peptídica , Ligação Proteica , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismoAssuntos
Escherichia coli/metabolismo , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Proteínas Ribossômicas/metabolismo , Fracionamento Celular/métodos , Códon , Imunoglobulina G , Cinética , Métodos , Poli U/metabolismo , RNA de Transferência/metabolismo , RNA Viral/metabolismo , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/metabolismo , Ribossomos/ultraestruturaRESUMO
The ribosomal protein S1 was modified by reductive methylation of some of its lysyl ammonium groups (S1). With 6 out of 30 groups methylated the protein lost its capacity to form stable complexes with polyuridylate. Addition of excess polyuridylate inhibited the methylation of the lysyl groups. In equilibrium dialysis experiments it was shown that the binding constant between S1 and U15 was lowered 10-fold as compared to the native protein. The pH-dependence of the complex formation between S1 and U15 confirms a participation of the lysyl residues. When S1 depleted 30-S ribosomes were reconstituted with methylated S1 these ribosomes were inactive in the poly(U) stimulated Phe-tRNA binding. The data are discussed with respect to a grid-like interaction between the lysyl groups of the protein and the phosphodiester bonds of the polynucleotide as a molecular basis of protein nucleic acid interaction.