Disruption of the Saccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin.

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.

Expression of rat neuronal nitric oxide synthase in Saccharomyces cerevisiae.

The rat neuronal nitric oxide synthase (nNOS) cDNA was expressed in Saccharomyces cerevisiae under the control of a hybrid PGK/GAL promoter, PAL. Galactose induction resulted in the production of a soluble 160 kDa protein that was recognised by anti-neuronal NOS antibody. NOS activity was detected by the conversion of [3H]arginine to [3H]citrulline and required the addition of the cofactors, calmodulin and tetrahydrobiopterin. The activity was inhibited by the arginine analogues NG-nitro-L-arginine and NG-nitro-L-arginine methyl ester.

Purification and characterisation of an Aspergillus niger invertase and its DNA sequence.

A secreted invertase was purified 23-fold by ultrafiltration, ion-exchange, and gel filtration chromatography from the culture supernatant of 18 h sucrose-grown cultures of Aspergillus niger. The purified enzyme hydrolysed sucrose and raffinose but there was no detectable hydrolysis of inulin, melezitose or PNPG. Invertase activity was optimal at pH 5.5 and 50 degrees C. The molecular mass of reduced invertase was 115 kDa, as determined by SDS gel electrophoresis. The native molecular weight of between 225 kDa and 250 kDa, estimated by electrophoresis under non-denaturing conditions, suggests that the protein is a dimer of identical subunits. The suc1 gene encoding this protein was completely-sequenced. The translated sequence yields a protein of 566 amino acids with a calculated molecular mass of 61 kDa, suggesting that carbohydrates represent about 50% of the mass of the protein.

Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations.

The glucose oxidase gene (god) from Aspergillus niger was expressed in Hansenula polymorpha using the methanol oxidase promoter and transcription termination region and the MF-alpha leader sequence from Saccharomyces cerevisiae to direct secretion. The expression cassette was cloned into the S. cerevisiae vector YEp13 and used to transform H. polymorpha strain A16. In the initial transformants plasmid replication was unstable, but was stabilized by a growth regime consisting of alternating cycles of selective and non-selective growth. The stabilized strain was grown to high cell density by fed-batch fermentation. Upon induction of the MOX promoter, glucose oxidase synthesis was initiated. At the end of the fermentation, the culture density was 76 g dry weight/1 and 108 IU/ml (0.5 g/1 or 0.65% dry weight) glucose oxidase was found in the culture medium; a further 86 IU/ml (0.43 g/1 or 0.56% dry weight) was recovered from the cell lysate. A plate assay was used to monitor glucose oxidase levels in individual colonies. This was then used to isolate mutants which showed abnormal regulation of god expression or which showed an altered pattern of secretion. One mutant, which showed increased production of glucose oxidase, was grown to high cell density by fed-batch fermentation (100.6 g/l) and produced 445 IU/ml(2.25 g/l or 2.2% dry weight) extracellularly and 76 IU/ml (0.38 g/l or 0.4% dry weight) intracellularly. The mutant thus not only increased total production but exported 83% of the total enzyme made compared to 55% in the parent strain.

A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor.

The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.

Characterization of the glyoxysomal isocitrate lyase genes of Aspergillus nidulans (acuD) and Neurospora crassa (acu-3).

The nucleotide sequences of the genes encoding the acetate-inducible glyoxylate cycle enzyme isocitrate lyase from the ascomycete fungi Aspergillus nidulans (acuD) and Neurospora crassa (acu-3) are presented. The respective A. nidulans and N. crassa genes are interrupted at identical positions by two introns and encode proteins of 538 and 543 amino acids, which have 75% identity. The predicted protein sequences do not demonstrate the C-terminal tripeptide S-K-L that has been implicated in peroxisomal targeting and found in the glyoxysomally located enzyme malate synthase from the same species. However, the protein sequences do exhibit a partial repeat which, in common with malate synthase, is located in regions that are absent from, or non-homologous with, the E. coli enzyme, which is not compartmentalized.

Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae.

Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to greater than 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 x 10(6) M-1 s-1, and for two-chain tPA, 0.7 x 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.

Saccharomyces cerevisiae strains that overexpress heterologous proteins.

We describe a system that facilitates the selection of host mutants that overproduce a range of secreted and internally produced heterologous proteins in Saccharomyces cerevisiae. These mutants were initially selected for their ability to oversecrete recombinant human albumin (rHA), as detected by a direct visual assay that relies upon antibody precipitation in solid media. Yeast strains that were able to synthesize and secrete increased levels of rHA also produced elevated levels of internally expressed proteins including alpha 1-antitrypsin Pittsburgh variant and plasminogen activator inhibitor type 2.

Expression of the Aspergillus niger glucose oxidase gene in A. niger, A. nidulans and Saccharomyces cerevisiae.

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.

Sequence and centromere proximal location of a transformation enhancing fragment ans1 from Aspergillus nidulans.

The Aspergillus nidulans sequence ans1, previously known to enhance transformation frequencies of pyr4-based vectors, was shown to enhance the efficiency of argB and trpC-based vectors. Increased efficiencies could be obtained by constructing vectors containing argB and ans1 or by cotransforming selectable plasmids (containing argB, trpC, or pyr4) with the non-selectable ans1 sequence. The preponderance of evidence suggests that the mechanism of ans1 activity does not involve homologous recombination events, in spite of the presence of multiple regions of homology in the A. nidulans genome. Genetic mapping localized ans1 to the vicinity of the centromere of linkage group I. The nucleotide sequence of a 1.8 Kb functional subclone of ans1 was determined and found to be highly A + T rich (81%).

Gene cloning in Aspergillus nidulans: isolation of the isocitrate lyase gene (acuD).

An Aspergillus nidulans gene library was constructed in a high-frequency transformation vector, pDJB3, based on the Neurospora crassa pyr4 gene. This gene library was used to isolate the structural gene for isocitrate lyase (acuD) by complementation of a deficiency mutation following transformation of A. nidulans. Plasmids rescued in Escherichia coli were able to transform five different A. nidulans acuD mutants. Transformation using plasmids containing the cloned fragment resulted in integration at the acuD locus in six of nine transformants.

Development of a high-frequency transforming vector for Aspergillus nidulans.

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.

Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa.

Relief of an auxotrophic requirement for uridine in Aspergillus nidulans strain G191 has been achieved by transformation with a segment of Neurospora crassa DNA containing the corresponding gene coding for orotidine-5'-phosphate decarboxylase. The mitotic stability of such transformants suggests that the DNA has integrated into the genome. Southern hybridisation analysis of DNA isolated from transformants revealed the presence of pBR322 sequences which have integrated into the host genome along with the N. crassa DNA.