Showing papers on "Pichia pastoris published in 1991"

High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene.

Abstract: We have used the methylotrophic yeast, Pichia pastoris, to express high levels of tetanus toxin fragment C, a potential subunit vaccine against tetanus. In high biomass fermentations fragment C was induced to 27% of total cell protein or about 12 g/l of culture. The purified protein was as effective as native fragment C in immunizing mice. In order to optimize fragment C production, we have examined the parameters affecting foreign gene expression in Pichia. The level of expression was found to be largely independent of the site of chromosomal integration of the gene (AOX1 or HIS4), the type of integrant (insertion or transplacement), and the methanol utilisation phenotype of the host strain (Mut+ or Muts). The most important factor in obtaining high levels was the presence of multiple integrated copies of the fragment C expression cassette. Multicopy clones could be isolated from transformations using DNA fragments targeted for single-copy transplacement into the chromosome. These multicopy transformants were surprisingly stable over multiple generations during growth and induction in high cell density fermentations. Analysis of chromosomal DNA from these clones suggests that they arose by circularization of the transforming DNA fragment in vivo followed by multiple insertion into the chromosome via repeated single crossover recombination, in addition to the expected transplacement event. We have found this to be a general phenomenon and have used these multicopy "transplacement" clones to obtain high-level expression of several other foreign genes in Pichia.

Yeast systems for the commercial production of heterologous proteins.

Abstract: Yeasts are attractive hosts for the production of heterologous proteins. Unlike prokaryotic systems, their eukaryotic subcellular organization enables them to carry out many of the post-translational folding, processing and modification events required to produce "authentic" and bioactive mammalian proteins. In addition, they retain the advantages of a unicellular microorganism, with respect to rapid growth and ease of genetic manipulation. The vast majority of yeast expression work has focused on the well-characterized baker's yeast Saccharomyces cerevisiae. However, with the development of DNA transformation technologies, a growing number of non-Saccharomyces yeasts are becoming available as hosts for recombinant polypeptide production. These include Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, Schwanniomyces occidentalis and Yarrowia lipolytica. The performance of these alternative yeast expression systems is reviewed here relative to S. cerevisiae, and the advantages and limitations of these systems are discussed.

Production of insulin-like growth factor-1 in methylotrophic yeast cells

Abstract: Insulin-like growth factor-1 (IGF-1), a naturally occurring, relatively short, single chain polypeptide, is prepared by growing methylotrophic yeast transformants containing in their genome at least one copy of DNA encoding IGF-1, in operational linkage with DNA encoding a signal sequence, which is effective for directing secretion of proteins from the host cells and which also includes the proteolytic processing site lys-arg and may include one or more glu-ala sequences. In preferred embodiments the signal sequence is the S. cerevisiae alpha mating factor pre-pro sequence. Expression of both the DNA encoding IGF-1 and the pre-pro signal sequence is regulated by a promoter region derived from a methanol responsive gene of a methylotrophic yeast. DNA constructs and recombinant methylotrophic yeast strains used for the expression and secretion of IGF-1 are also provided. For preferred embodiments, protease deficient Pichia pastoris strains are provided.

High-level secretion of biologically active aprotinin from the yeast Pichia pastoris.

Abstract: A synthetic gene encoding aprotinin (bovine pancreatic trypsin, inhibitor) was fused to theSaccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing sitePichia pastoris strains were developed to'express one or multiple copies of a methanol-inducible expression cassette containing the gene fusionP pastoris containing a single copy of the vector secreed approximately 150 mg/l of immunoreactive protein A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin The purified aprotinin molecule was equipoten with the native molecule in a trypsin inhibition assay Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor The N-terminal extension was variably 11 or 4 amino acids Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension

Method for the expression of heterologous genes in the yeast Pichia pastoris, expression vectors and transformed microorganisms

Abstract: The present invention provides a highly efficient method for the expression of heterologous genes, using as the host an auxotrophic his3 mutant of strain BKM-90 of Pichia pastoris and as recombinant DNA for the transformation thereof an integrative vector which contains a functional selection marker of Saccharomyces cerevisiae , the promotor of the alcohol oxidase gene (AOX₁) of the yeast itself, as the transcription terminator that of glyceraldehyde 3-phosphate dehydrogenase (GAP) of S . cerevisiae , or that which is carried by the heterologous gene to be expressed, and another sequence homologous to the genome of Pichia pastoris which serves for the integration thereof. The expression vectors used further contain a heterologous gene bound to the signal peptide of the sucrose invertase gene (SUC2) of S . cerevisiae . This method guarantees that the polypeptide products are recoverable under controllable conditions and with high yields. The strains according to the present invention can be used for the large-scale production of pharmaceutical products or of enzymes of industrial interest.

Method for obtaining recombinant surface antigen of hepatitis B virus (HEP B) of higher immunogenic capacity and use thereof in a vaccine preparation

Abstract: A multi-step process for recovering hepatitis B surface antigen from Pichia pastoris cells containing a gene encoding said hepatitis B surface antigen and having expressed it. The process leads to a high yield of very pure, homogeneous HBsAg in a particulated form having a high immunogenicity, suitable for use in vaccine compositions for treating human beings and for use in diagnostic methods.

Biological production of acetaldehyde from ethanol using non-growing Pichia pastoris whole cells

Abstract: Acetaldehyde has been produced biologically using whole-cellPichia pastoris in a semibatch fermentor. Ethanol and air were fed continuously, and the product, acetaldehyde, was removed by the air stream. Operation of the reactor exceeded 100 h, maintaining high alcohol oxidase activity. Low cell-mass concentration (9.9 g/L) minimized product inhibition. Ethanol concentration in the broth, oxygen concentration in the air, and pH were evaluated for their effects on the fermentation process.

Pichia pastoris acid phosphatase gene

Abstract: The present invention discloses the Pichia pastoris acid phosphatase gene, which includes the 5' regulatory region, signal sequence, structural gene, and 3' transcription termination sequence. Also disclosed are methods of using these fragments, which include but are not limited to the secretion of proteins from cells and the regulation of the transcription of DNA. DNA vectors containing the acid phosphatase gene or fragments thereof and hosts transformed with these vectors are also disclosed. Additionally, integrative vectors which direct integration at the Pichia pastoris PHO1 locus and a method of identifying these disruptants is disclosed.

Drosophila glucose dehydrogenase and yeast alcohol oxidase are homologous and share N-terminal homology with other flavoenzymes

Abstract: The presence of similar nucleotide cofactor-binding domains in otherwise unrelated enzymes has been rationalized on the basis of the exon-shuffling model of protein evolution (Gilbert 1978). In the common case of enzymes which share a similar nucleotide-binding domain but exhibit different specificities for primary substrates, the degree of sequence similarity is restricted to small regions specifically demonstrated to bind to the nucleotide cofactor. This pattern of restricted similarity suggests that dissimilar regions arose from nonhomologous exons. We show herein that the sequence similarity of two unrelated flavoenzymes, Drosophila glucose dehydrogenase ( GLD ) and yeast alcohol/methanol oxidase ( AOX / MOX ) are unexpectedly similar throughout their primary sequence. In addition these enzymes share considerable similarity both with other unrelated flavoenzymes and with the ras protein for a small amino-terminal region encoding their nucleotide-binding sites. The genes encoding GLD (E.C. 1.1.99.10) and AOX/MOX (E.C. 1.1.3.13) recently have been isolated from fruit flies (Cavener et al. 1986~; Krasney et al. 1990) and yeast (Ledeboer et al. 1985; Koutz et al. 1989), respectively. The biochemical properties of Drosophila GLD are very similar to those of Aspergillus oryzue GLD, which catalyzes the oxidation of D-glucose to D-glucono-l Cavener et al. 1986b). AOX/MOX is associated with the ability of four yeast genera to utilize methanol as the sole carbon source (Lee and Komagata 1980). It catalyzes the oxidation/reduction of methanol and oxygen to formaldehyde and hydrogen peroxide, respectively. Thus, GLD and AOX/MOX exhibit remarkably different biochemical and functional properties. A search of both the National Biomedical Research foundation (NBRF) protein data base and the GenBank nucleic acid data base unexpectedly revealed a significant degree of similarity between D. melanogaster GLD and Hansenula polymorpha (yeast) MOX and Pichia pastoris (yeast) AOX 1. The statistical significance was evaluated by comparing the similarity of the primary amino acid sequence of GLD with 200 randomized sequences of AOXl and MOX by using the RDF2 program of W. Pearson (University of Virginia). The randomized sequences were of the same length and amino acid composition as AOXI and MOX. The optimized alignment scores for the comparison of AOX 1 with GLD and for the comparison of MOX with GLD were greater than 30 SD above the mean of the randomized scores and are more than threefold higher than those of the highest random sequence. Percentage similarities

Oxygen and temperature effects on acetaldehyde-induced catabolite inactivation in Pichia pastoris

Abstract: Exposure of methylotrophic yeasts to other carbon sources after growth on methanol results in catabolite inactivation. As a result, peroxisomes are rapidly degraded effectively disabling the metabolic pathway initiated by alcohol oxidase in favour of a more energetically favourable route. A model equation has been developed to describe the effect of temperature, dissolved oxygen concentration and acetaldehyde (catabolite) concentration on catabolite inactivation in Pichia pastoris. When pre-exposed to 4 g/l acetaldehyde at 30°C, the rate of conversion of ethanol to acetaldehyde decreased by 75%. Inactivation was reduced to 45% at 30°C by reducing the dissolved oxygen concentration. At high dissolved oxygen concentration, enzyme function was only inactivated by 20% if the temperature during the period of exposure to acetaldehyde was reduced to 5°C. The influence of acetaldehyde can be eliminated completely by operating at 5°C and low dissolved oxygen concentrations. Application of these findings to process design has enabled us to conduct preliminary reactions in laboratory-scale reactors that have yielded acetaldehyde concentrations greater than 3 M (130 g/l) in 4 h.

Catalase-negative pichia pastoris

Abstract: A novel strain of P. pastoris and an alcohol oxidase free of catalase activity produced therefrom is provided.

Serum or antibody resistant to yeast, their preparation and their employment

Abstract: PURPOSE: To obtain the subject antibody used for the purification of a B type hepatitis vaccine produced from a yeast as a host by immunizing a vertebrate with the yeast or its fragment to prepare an astisera recognizing the yeast, and subsequently purifying the antisera to give an immunoglobulin fraction. CONSTITUTION: A yeast (e.g. Pichia pastoris) or its fragment as an antigen is intravenously injected into a vertebrate (e.g. rabbit) by the use of an injector for the immunization of the vertebrate. The same operation are repeated every three days after the first injection, and after 24 days, the whole blood of the rabbit is collected. The blood is centrifuged to give the serum, from which an antisera recognizing the yeast is prepared. The antisera is subjected to an ammonium sulfate fractionation treatment, and the obtained IgG fraction (immunoglobulin G) is applied to a column filled with a carrier bonded to protein A, etc. The adsorbed fraction is eluted to obtain the objective antibody specifically recognizing the yeast. COPYRIGHT: (C)1993,JPO&Japio

Method for obtaining recombinant surface antigen of hepatitis B virus (HEP B)

Abstract: of EP0480525A multi-step process for recovering hepatitis B surface antigen from Pichia pastoris cells containing a gene encoding said hepatitis B surface antigen and having expressed it. The process leads to a high yield of very pure, homogeneous HBsAg in a particulated form having a high immunogenicity, suitable for use in vaccine compositions for treating human beings and for use in diagnostic methods.