ABSTRACT
Expression of recombinant human albumin (rHA) in Saccharomyces cerevisiae resulted in secretion of both mature albumin and a 45 kDa degradation product, comprising an N-terminal fragment of rHA with heterogeneous C-termini between residues 403 and 409 (Geisow et al., 1991). Site-directed mutagenesis of the human albumin gene (HA) to change Arg410 to Ala (R410A) caused a significant reduction in the amount of fragment produced. Mutation of the adjacent dibasic site Lys413 Lys414 had little effect in isolation, but in combination with the R410A mutation resulted in a further reduction in the amount of rHA fragment produced. This reduction could be duplicated with nature-identical rHA by disruption of the gene for an aspartyl protease (YAP3), alone or in conjunction with disruption of the KEX2 gene. Disruption of KEX2 alone did not result in any improvement in the degree of degradation of the rHA. Reduced degradation was also observed when an rHA-human growth hormone fusion protein was secreted from a yap3 strain, suggesting that such strains may have a general utility for heterologous protein secretion.
Subject(s)
Albumins/metabolism , Aspartic Acid Endopeptidases/genetics , Proprotein Convertases , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Albumins/genetics , Aspartic Acid Endopeptidases/metabolism , Blotting, Western , Densitometry , Electrophoresis, Polyacrylamide Gel , Gene Expression , Humans , Mutagenesis, Site-Directed , Plasmids/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Subtilisins/genetics , Subtilisins/metabolismABSTRACT
We report the cloning of the Aspergillus niger glucose oxidase gene and its use to elevate glucose oxidase productivity in A. niger by increasing the gene dosage. In addition, the gene has been introduced into A. nidulans where it provides the novel capacity to produce glucose oxidase. A plasmid, in which DNA encoding the mature form of glucose oxidase was preceded by a Saccharomyces cerevisiae secretion signal, effected high-level production of extracellular glucose oxidase in this yeast.
Subject(s)
Aspergillus nidulans/genetics , Aspergillus niger/genetics , Genes, Fungal , Glucose Oxidase/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Aspergillus niger/enzymology , Base Sequence , Blotting, Western , Cloning, Molecular , DNA, Fungal , Gene Expression , Glucose Oxidase/metabolism , Molecular Sequence Data , Plasmids , Restriction MappingSubject(s)
Actinomycetales , Bacterial Infections/microbiology , Opportunistic Infections/microbiology , Actinomycetales/classification , Actinomycetales/drug effects , Actinomycetales/genetics , Actinomycetales/pathogenicity , Drug Resistance, Microbial , Humans , Immune Tolerance , Sepsis/microbiologyABSTRACT
Investigation of 39 JK-type coryneform isolates from patients at a single hospital revealed that 23 possessed plasmids, which formed six groups on restriction endonuclease analysis. Four of the groups were associated with production of similar bacteriocin-like substances, and shared a minimum of 6.4 kilobase pairs of DNA. These plasmids, found in isolates from different patients, provide strong direct evidence that person-to-person transmission of JK bacteria had occurred within the hospital.
