EP0313584A1 - Polypeptides stabilises ainsi que leur expression et utilisation en fermentation - Google Patents

Polypeptides stabilises ainsi que leur expression et utilisation en fermentation

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Publication number
EP0313584A1
EP0313584A1 EP19870905632 EP87905632A EP0313584A1 EP 0313584 A1 EP0313584 A1 EP 0313584A1 EP 19870905632 EP19870905632 EP 19870905632 EP 87905632 A EP87905632 A EP 87905632A EP 0313584 A1 EP0313584 A1 EP 0313584A1
Authority
EP
European Patent Office
Prior art keywords
yeast
polypeptide
glycosylated
glycosylation
plasmid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19870905632
Other languages
German (de)
English (en)
Inventor
William Edward Lancashire
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Whitbread PLC
Original Assignee
Whitbread PLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Whitbread PLC filed Critical Whitbread PLC
Publication of EP0313584A1 publication Critical patent/EP0313584A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)

Definitions

  • the present invention relates to polypeptides which don been modifief to infer increased stability, and to the genetic modification of yeasts which then both express and secrete such polypeptides in a brewing or other fermentation process.
  • BACKGROUND OF THE INVENTION Yeasts secrete polypeptides, and can be modified to secrete foreign polypeptides owing to the presence of a genetic secretion signal, e.g. the prepro-region of the " ⁇ -factor" sequence. Secretion may be accompanied by glycosylation if the polypeptide includes the "trigger" sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino-acid. Mannose in the cell is used to build up glycosyl groups.
  • the enzyme can be expressed in and secreted from yeast after hybridisation to the above-mentioned secretion signal, both at the DNA level.
  • the enzyme contains a suitable trigger sequence, because it is glycosylated on secretion.
  • EP-A-0171000 discloses a method of producing a protein or peptide, which comprises culturing yeast transformant immobilised on a carrier gel, the transformant being transformed by a secretor expression vector.
  • the secretion factor ensures that a desired polypeptide can be secreted from yeast.
  • the polypeptide may contain one or more glyccsylation signals, but the polypeptide which is produced may not be glycosylated if, as seems likely, any glycosylation signal is usually cleaved. For example, the photograph reproduced as Figure 11 does not suggest that IL-2 has been glycosylated.
  • WO-A-8603777 discloses that the pre-region of premelibiase can be used in engineering secretion, from genetically-modified yeast, or peptides and proteins other than melibiase.
  • EP-A-0201208 published 12.11.86, i.e. after the priority date of 01.09.86 claimed in this application discloses "supersecreting" mutants of Saccharomyces cerevisiae.
  • the mature protein is that actually secreted by the cell, by "processing" a precursor polypeptide or protein, as synthesised within a cell, having a signal sequence attached to the mature form. The purpose or practice of this oligosaccharide addition is unclear.
  • the gene for encoding bacterial ⁇ -lactamase occurs naturally and is found on many of the currently-used cloning vectors, as a selectable marker.
  • the gene confers en the host resistance to ampicillin and other penicillin derivatives.
  • the gene When introduced into yeast by transformation, the gene is weakly expressed from its own promoter; it has been expressed at much higher levels by using various yeast promoters to drive the expression (Hollenberg, C.P. (1982) Current Topics Microbiol. Immunol., 96 119-144).
  • a glycosylated analogue of a selected polypeptide containing no glycosylation signal is produced by expression and secretion from yeast in a fermentation process; to achieve this end, DNA encoding a glycosylation signal and (if necessary) a secretion signal is inserted into the organism which is then cultured. It is likely that the glycosylated analogue has the same activity and superior stability.
  • the (non-glycosylated) selected polypeptide may be naturally secreted from the organism, i.e. a secretion signal (which is usually cleaved on secretion) is encoded and expressed. If it is not, the insertion must also encode the secretion signal.
  • a secretion signal which is usually cleaved on secretion
  • Brewer's or distiller's yeast may be a natural host for expression of the selected polypeptide.
  • the invention can also be applied if the polypeptide is foreign to the yeast; in this case, the insertion must also encode the polypeptide.
  • a "selected" polypeptide encoded by yeast DNA is modified in up to three ways.
  • the structure of the expressed polypeptide includes a secretion signal, e.g. the signal region of ⁇ -factor when the host is to be S. cerevisiae.
  • the structure as expressed includes a glycosylation signal, e.g. an amino-acid triplet as given above.
  • the glycosylation signal e.g. an amino-acid triplet as given above.
  • secretion it is glycosylated. All of these three factors (or as many of them as are required) can be organised so that the function of the selected polypeptide is effectively unchanged, while glycosylation provides stability.
  • the genetic information encoding the glycosylated polypeptide may be carried on a plasmid vector.
  • a plasmid e.g. a circular plasmid, for use in the invention, comprises a first sequence which allows for growth in a yeast in cell culture and a second sequence which codes for a polypeptide including both a eukaryotic secretion signal and a eukaryotic glycosylation signal.
  • a plasmid may be cloned, and maintained in a suitable host, e.g. E. coli, as desired.
  • a plasmid for use in the invention may be especially constructed to take account of the stability which has been found for glycosylated polypeptides.
  • the genetic information in the plasmid may be the same as that in known vectors which have*been modified to encode a secretion factor and in which the glycosylation factor is part of the structure of the expressed polypeptide.
  • polypeptides e.g. the bacterial enzyme ⁇ -glucanase
  • ⁇ -glucanase are glycosylated naturally on secretion from the unnatural host S. cerevisiae.
  • the information encoding the glycosylated polypeptide is stably integrated into the yeast chromosome. Suitable techniques are described in EP-A-0231608.
  • the selected polypeptide may be any polypeptide having a desired function in the brewing process, e.g. a non-naturally-occurring protein such as a hybrid enzyme or other semi-synthetic protein, or a naturally-occurring enzyme.
  • the gene for the polypeptide can be isolated by a conventional procedure, cloned (if desired), and inserted into a plasmid for use in the invention or otherwise into the DNA of the vector.
  • conventional site-directed mutagenesis may be used. Site-directed mutagenesis should minimise structural changes to the selected polypeptide.
  • Site-directed mutagenesis is a technology well-documented by Eckstein and co-workers (Taylor et al, Nucleic Acids Res. 13, 8749-8785).
  • the point mutation required may be determined by the use of a synthetic oligonucleotide spanning the region to be mutated. This oligonucleotide is designed to have a mismatch as required. Shown beloware the region of interest in the mature ⁇ -lactamase gene, the mutated region in the ⁇ -lactamase product, and the corresponding oligonucleotide (which may be made synthetically) required in the procedure to generate the 'glycosylation' site:
  • a host containing plasmid containing the ⁇ -lactamase region may be subjected to site-directed mutagenesis, using the synthetic oligonucleotide and reagents and procedures in a commercially-available kit (Amersham system code RPN 2322), Colonies may then be chosen and the vectors contained therein screened by DNA sequencing, e.g. using the dideoxy chain termination method.
  • the complete ⁇ -lactamase gene is then constructed and the modified gene is introduced into yeast. For stability studies, it is necessary to incorporate on to the plasmid a yeast DNA origin of replication and a selectable marker.
  • the invention is not limited to the objective of protection or stabilisation against protease-degraded polypeptides.
  • the invention can be used for the production of a glycosylated analogue of, say, ⁇ -amylase, a foam-stabilising protein, ⁇ -amylase, pullulanase, amylcglucosidase or any other peptide of industrial significance or value, e.g. a pharmaceutically-active peptide.
  • Example 1 illustrates the invention, using as a model bacterial ⁇ -lactamase.
  • the Example illustrates cloning of an oligonucleotide containing the glycosylation site on to the gene, either at the terminus of the enzyme coding region or within the coding region itself.
  • GAL UAS Upstream Activation Sequence from yeast gene
  • GAL Tc R tegracycline resistance gene
  • ORI E. coli origin of replication
  • S secretion signal of ⁇ -lactamase
  • O oligonucleotide
  • the starting plasmid was pCLAlOl (see Figure 1; also disclosed by A. Mileham et al., in preparation).
  • This bacterial plasmid contains the ⁇ -lactamase expression system. Expression of the ⁇ -lactamase is under the control of the yeast CYC1 gene promoter which in turn is regulated by the Upstream Activation Sequence taken from the yeast GAL1/GAL10 gene. The latter permits gene expression to be turned on by growth in galactose-containing medium.
  • the ⁇ -lactamase has been modified such that a recognition site for the restriction endonuclease Bglll has been introduced to coincide with the point at which the secretion signal is cleaved to produce the mature enzyme.
  • This site is used here to insert the glycosylation site-containing oligonucleotide.
  • Escherichia coli strain 5K (thi-, thr-1, leuB6, lacY1, tonA21, supE44 r K - m K -), and selection of positive, colonies on Lauria Broth (LB) medium containing 50 (ig/ml tetracycline, clones were screened by the Grunstein & Hogness colony hybridisation technique (Proc. Natl. Acad. Sci. (1975) USA 72, 3961-3965) using the above oligonucleotide as a probe after kinase-labelling with 32 P-gamma-adenosine triphophate (Maniatis (1982) A Laboratory Manual CSH, page 123). Positive colonies identified after X-ray film exposure were then isolated and plasmid DNA prepared (Birnboim & Doly (1979) Nucleic Acids Res. 7 , 1513).
  • the correct orientation of insertion of oligonucleotide into the Bglll site is very important as the glycosylation signal must remain in frame with the ⁇ -lactamase enzyme coding region.
  • the orientation of the insertion in the clones was determined by the dideoxy DNA sequencing method. Several examples of the clones were each cleaved with the two restriction enzymes EcoRI and PstI, and the released fragment ligated into sequencing vector M13mp19 which had been similarly cleaved. One of the clones, named pCLA101-0, was identified as containing the oligonucleotide cloned in the required orientation.
  • yeast transformation In order to be able to introduce the modified gene into yeast for stability studies, it was necessary to incorporate a yeast DNA origin of replication and a selectable marker. This was achieved using standard genetic construction techniques (see Kaniatis (1982) A Laboratory Manual CSH). Plasmid YEp213 (see Fig. 2; Sherman et al. Methods in Yeast Genetics, Cold Spring Harbor, 1986) was used as a source of yeast replicon and selectable marker (LEU2 gene). The large Aatll-Sall fragment contains the required sequences and this was substituted for the Aatll-Sall fragment in pCLA101-0 by cleavage with Sail and Aat.II, and ligation. This product vector is called YEpCLA101-0 (see Fig. 3). Yeast transformation
  • the plasmid YEpCLA101-0 (approx. 1 ⁇ g) was transformed into yeast strain JRY188 ( ⁇ leu2-3, 122 his3 ⁇ 1 trpl-289 ura3-52 sir3-8) using the lithium salt mediated procedure (Ito et al. (1983) J. Bacteriol. 153, 163-168). Selection of plasmid containing transformants was made on synthetic medium lacking leucine. Cultures for ⁇ -lactamase studies
  • Two yeast strains were used for comparative studies. One was the transformant described above containing the newly constructed plasmid YEpCLA101-0 and the other was a control strain containing plasmid YEpCLA101. This latter plasmid is identical to YEpCLA101-0 except that the oligonucloeotide containing the glycosylation site is not present.
  • Polyacrylamide slab electrophoresis (native) was performed in 10% gels (Maurer (1971), Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, de Gruyter, Berlin, 44-45).
  • culture supernatants were concentrated 25-fold.
  • the gel was loaded with 20 ⁇ l sample per track. This volume contained approximately 25 mg total protein of which approximately one-third is ⁇ -lactamase. In the case of the endoH-treated samples, half this amount of protein was present.
  • track 1 is for untreated culture A (YEpCLA101-0 in JRY188) and track 2 is for supernatant concentrate C (YEpCLA101 i0 JRY188).
  • Tracks 3 and 4 are respectively for A+(endoH-treated culture supernatant concentrate) and A-(minus endoH, control culture supernatant concentrate).
  • Tracks 5 and 6 are respectively for C+(e(doH-treated culture supernatant concentrate) and C-(minus endoH, control culture supernatant control).
  • Comparison of tracks 1 and 2 shows that the introduction of the oligonucleotide has led to a considerable proportion of enzyme being glycosylated.
  • the activity in the smear at the top of the gel corresponds to high molecular weight glycosylated protein.
  • Comparison of tracks 3 and 4 shows that the high molecular weight smear is removed by the treatment, confirming it to be a glycosylated form of the enzyme. Stability studies
  • a primary purpose of this invention is to increase the stability of a foreign protein by deliberate introduction of glycosylation into the protein produced by yeast.
  • One test relevant to brewing which determines protein stability is the protein's resistance to or tolerance of proteolytic enzymes.
  • the effects of the enzyme trypsin on the above-mentioned enzyme preparations have been studied with a view to determining that stability in comparison to their non-glycosylated analogues.
  • results which are means of triplicate assays, are given in the following Table. They show that the glycosylated, modified enzyme is significantly more resistant to trypsin inactivation than the unmodified control.
  • activities are expressed in terms of arbitary spectrophotometer units per minute per OD 600 unit of original yeast culture.
  • the figures are percentages of activity remaining after treatment compared to the activities in the untreated samples.
  • the results demonstrate the effectiveness of the process in stabilising foreign proteins against proteolytic attack.

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Abstract

Dans un procédé de fermentation de levure, utilisé par exemple dans le domaine de la brasserie ou de la distillation, l'ADN de la levure est modifié de sorte qu'un polypeptide sélectionné, qui n'est pas sécrété naturellement par la levure sous une forme glycosylée, est exprimé et sécrété sous la forme d'un analogue glycosylé.
EP19870905632 1986-09-01 1987-09-01 Polypeptides stabilises ainsi que leur expression et utilisation en fermentation Pending EP0313584A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8621118 1986-09-01
GB868621118A GB8621118D0 (en) 1986-09-01 1986-09-01 Stabilised polypeptides

Publications (1)

Publication Number Publication Date
EP0313584A1 true EP0313584A1 (fr) 1989-05-03

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Family Applications (2)

Application Number Title Priority Date Filing Date
EP19870905632 Pending EP0313584A1 (fr) 1986-09-01 1987-09-01 Polypeptides stabilises ainsi que leur expression et utilisation en fermentation
EP87307715A Withdrawn EP0262805A1 (fr) 1986-09-01 1987-09-01 Polypeptides stabilisés et leur expression et utilisation dans la fermentation

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP87307715A Withdrawn EP0262805A1 (fr) 1986-09-01 1987-09-01 Polypeptides stabilisés et leur expression et utilisation dans la fermentation

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EP (2) EP0313584A1 (fr)
GB (1) GB8621118D0 (fr)
WO (1) WO1988001647A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5218092A (en) * 1988-09-29 1993-06-08 Kyowa Hakko Kogyo Co., Ltd. Modified granulocyte-colony stimulating factor polypeptide with added carbohydrate chains
DE3901917A1 (de) * 1989-01-24 1990-07-26 Behringwerke Ag Mutanten von humanem antithrombin iii
JP3318602B2 (ja) 1997-11-10 2002-08-26 独立行政法人産業技術総合研究所 糖鎖付加型ヘパリン結合性タンパク質、その製造方法およびそれを含有する医薬組成物
CN1325443A (zh) * 1998-10-30 2001-12-05 诺沃奇梅兹有限公司 具有减弱的变应原性的糖基化蛋白

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NO840200L (no) * 1983-01-28 1984-07-30 Cefus Corp Glukoamylase cdna.
EP0143081B1 (fr) * 1983-11-21 1989-08-23 Ciba-Geigy Ag Synthèse d'activateur du plasminogene tissulaire(TPA) par levure
JPS6156078A (ja) * 1984-07-27 1986-03-20 Suntory Ltd 酵母を宿主とする分泌発現ベクタ−
JPS61141890A (ja) * 1984-12-15 1986-06-28 Suntory Ltd アルコ−ルの製造法
EP0201208B1 (fr) * 1985-04-09 1992-07-15 Collaborative Research Inc. Mutantes de saccharomyces cerevisiae à hypersécrétion

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8801647A1 *

Also Published As

Publication number Publication date
GB8621118D0 (en) 1986-10-08
WO1988001647A1 (fr) 1988-03-10
EP0262805A1 (fr) 1988-04-06

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