EP0192679A4 - Hefeklonierungsvektoren. - Google Patents

Hefeklonierungsvektoren.

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Publication number
EP0192679A4
EP0192679A4 EP19850904174 EP85904174A EP0192679A4 EP 0192679 A4 EP0192679 A4 EP 0192679A4 EP 19850904174 EP19850904174 EP 19850904174 EP 85904174 A EP85904174 A EP 85904174A EP 0192679 A4 EP0192679 A4 EP 0192679A4
Authority
EP
European Patent Office
Prior art keywords
yeast
sequence
hcg
fragment
hormone
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.)
Withdrawn
Application number
EP19850904174
Other languages
English (en)
French (fr)
Other versions
EP0192679A1 (de
Inventor
Anton K Beck
Gregory P Thill
Edward G Bernstine
Jeffrey F Lemontt
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.)
Applied Research Systems ARS Holding NV
Original Assignee
Integrated Genetics Inc
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 Integrated Genetics Inc filed Critical Integrated Genetics Inc
Publication of EP0192679A1 publication Critical patent/EP0192679A1/de
Publication of EP0192679A4 publication Critical patent/EP0192679A4/de
Withdrawn legal-status Critical Current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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]

Definitions

  • YEAST CLONING VEHICLES Background of the Invention This invention relates to cloning vehicles for production of proteins.
  • proteins The term “proteins” is used in this application to include peptides of indefinite size.
  • eukaryotic cells such as yeasts
  • cDNA complementary DNA
  • proteins exported from eukaryotic cells are processed in a secretory pathway that involves synthesis of a precursor protein (containing an amino-terminal "signal" peptide region), translocation across endoplasmic reticulum membranes, followed by specific signal peptide cleavage and further processing including carbohydrate additions (glycosylation) , and finally secretion of the mature product out of the cell.
  • the yeast Saccharomyces cerevisiae is known to have such a secretory pathway [see Schek an and Novick, "The secretory process and yeast cell-surface assembly", in The Molecular 3iology of the Yeast Saccharomyces; Metabolism and Gene
  • yeast secretion mechanism provides components capable of glycosylating proteins.
  • Hitzeman et al. (1983) Science 219;620-625 report expression and secretion in yeast of human interferon based on a coding region containing the inteferon signal sequence. The mature secreted protein reported by Hitzeman et al. demonstrated inaccurate cleavage of the signal peptide.
  • Hinnen et al. at an International Congress of Microbiology (Boston, Massachusetts 1982) reported the fusion of a DNA fragment from the PH05 gene of the yeast Saccharomyces cerevisiae (containing the promoter and 80% of the 5' end of the PH05 signal sequence) to a cDNA fragment coding for the mature protein sequence and a portion of the 3' e'nd of the signal sequence of human alpha interferon.
  • PH05 codes for the major repressible (by phosphate) acid phosphatase, a secreted enzyme in this yeast.
  • This construction lacks the DNA coding sequence for the last 3 amino acids of the PH05 signal peptide and instead the last three amino acids of the hybrid signal are those coded for by the human cDNA sequence—i.e., the amino acids of the pre-interferon signal information.
  • the plasmid constructed by Hinnen et al. was used to transform cells of Saccharomyces cerevisiae. The transformants were reported to express interferon activity under phosphate regulation. Localization of the interferon activity (intracellular versus extracellular) was not discussed.
  • the invention features cloning vehicles capable of effecting the expression in a yeast host of the DNA sequences coding for a' uman fertility hormone selected from the and 3 subunits.of human chorionic- gonadotropin (hCG) , luteinizing hormone (LH) , and follicle stimulating hormone (FSH) , the cloning vehicles including, in phase and in order of transcription, a yeast promoter sequence and a DNA sequence encoding the immature hormone subunit, including the signal peptide.
  • the most preferred hormones are ⁇ -hCG and ⁇ -hCG.
  • the promoter is substantially identical to the promoter sequence of a yeast PH05 gene, which preferably is within a 0.55 kb sequence upstream from the translational start site of the PH05 coding sequence contained within the 8 kb EcoRI PH05 genomic DNA fragment from chromosome 2 of Saccharomyces cerevisiae.
  • the promoter is substantially identical to the promoter sequence of a yeast GAPD gene, preferably within a 0.6 kb sequence of the 2,1 kb Hindlll GAPD genomic DNA fragment of Saccharomyces cerevisiae.
  • the GAPD gene encodes one species of glyceraldehyde-3'-phosphate alhydrogenase, a cytoplas i ⁇ (i.e., nonsecreted) enzyme in Syccharomyces cerevisiae.
  • Fig. 1 is a diagram representing the PH05 expression vector.
  • Fig. 2 is a diagram representing the GAPD expression vector.
  • Fig. 3 is a diagram representing the PH05- ⁇
  • Fig. 4 is a diagram representing the GAPD- ⁇ (A) and GAPD- 3 (B) cloning vectors.
  • PH05 Expression Vector Structure and Construction PH05 expression vector pl25N (Fig. 1) includes the following DNA segments (counterclockwise from the EcoRI site) :
  • TRP1 gene from yeast contained within a 1.45 kb EcoRI genomic DNA fragment from chromosome 4, described in Kingsman et al. (1979) Gene 1_, 141-152 and in Tschumper et al. (1980) Gene 10, 157-166.
  • This fragment contains a 103 bp functional TRP1 promoter region, a 672 bp coding sequence, and a 678 bp 3' untranslated region, which functions not only as a transcription termination sequence but also as a weak DNA replication origin (or replicon) called the arsl sequence;
  • plasmid YRp7 has been described by Tschumper et al., i ⁇ ⁇ r (1980) and consists of this 1.45 kb EcoRI fragment inserted into pBR322 at the EcoRI site in an orientation.such that TRP1 and the gene for ampicillin resistance are transcribed in the same direction; 2.
  • the 1.45 kb Hindi fragment from the B form of the 2 micron circle a naturally occurring plasmid endogenous to most strains of Saccharomyces cerevisiae, described in Broach, The Molecular Biology of the Yeast Saccaromyces; Life Cycle and Inher tance, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1981.
  • the Hindi fragment contains a "strong" origin of DNA replication, (i.e., “stronger” than arsl because it confers a higher plasmid copy number in yeast) and confers a much lower rate of plasmid loss per mitotic cell division. This is also partly due to the presence of endogenous 2-micron plasmids carried in most strains of yeast.
  • the orientation of the vector fragment in plasmid pl25N is not important for this function;
  • the PH05 expression vector pl25N is constructed using the conventional recombinant DNA procedures described in Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982. Plasmid YRp7 is partially digested with EcoRI such that only one of the two EcoRI sites is cleaved. Most of fhe product of this digestion consists of 5.8 kb linearized YRp7 molecules. Approximately half of these linear molecules are cleaved at one EcoRI site while the other half are cleaved at the other EcoRI site..
  • This mixture of linear molecules is separated from uncleaved circular molecules and from shorter linear molecules, arising by occasional cleavage of both EcoRI sites, by gel electrophoresis, as described in Maniatis et al. (1982) , followed by elution from the gel.
  • the 5' protruding EcoRI ends are then filled in with dNTP's using DNA polymerase I (Klenow enzyme).
  • DNA polymerase I Klenow enzyme
  • the 5' EcoRI protruding ends can first be removed with a single-strand-specific 5' to 3' exonuclease.
  • the flush-ended molecules thus generated are rejoined (circularized) with DNA ligase, which results in the loss of one of the EcoRI recognition sequences.
  • the plasmid of interest, YRp7' which retains the EcoRI site adjacent the TRP1 promoter, is identified by restriction mapping.
  • the 1.45 kb Hindi fragment from the yeast 2 micron cirjcle is ligated into the Nrul site of YRp7'.
  • the plasmid that is generated, YRp7'N is then digested with both EcoRI and BamHI.
  • the resulting large fragment, 6.87 Eb which is isolated by gel electrophorsis, is used to make pl25N, as described below.
  • the other major component of pl25N is the PH05 promoter.
  • the PH05 gene of Saccharomyces cerevisiae codes for the major phosphate-repressible acid phosphatase enzyme (APase), corresponding to the peptide called p60, and is contained within an 8 kb EcoRI genomic DNA fragment from chromosome 2 described in Bostian et al. (1980) PNAS 77, 4504-4508 and in Kramer and Andersen (1980) PNAS 11_, 6541-6545.
  • APIase major phosphate-repressible acid phosphatase enzyme
  • the PH05 promoter lies within the approximately 0.55 kb sequence that preceeds the ATG translational start triplet at the beginning sequence of the PH05 coding sequence and is excised and isolated from the PH05 gene as a DNA fragment using Kpnl endonuclease. (These Kpnl sites occur 1400 bp upstream (i.e. on the 5 1 side) of the ATG triplet and 51 bp downstream (i.e. on the 3' side) of the ATG triplet.
  • this Kpnl fragment is digested with Bal-31 exonuclease, such that approximately fifty to ninety base pairs from either end of the DNA fragment are cleaved. Digestion with Bal-31 exonuclease generates a series of fragments of varying lengths. These fragments are combined with DNA polymerase I (Klenow enzyme) to assure that all the molecules are blunt-ended, after which EcoRI linkers are added to the blunt-ended fragments and ligated to both ends with T4 DNA ligase.
  • DNA polymerase I Klenow enzyme
  • Plasmid pl25N contains the longer of the two fragments and includes, downstream from the PH05 promoter, the major PHOS start site. Plasmid plllN contains a shorter fragment which does not include the major PH05 -start site. pl25N and plllN were used to transform E.coli and transformants selected on the basis of ampicillin resistance. Plasmids pl25N and plllN, isolated from transformants, have been deposited in the NRRL, Peoria, IL, and given NRRL number B-15805 and B-15804, respectively.
  • plasmid pBGAP is an expression vector containing the promoter from the major gene encoding glyceraldehyde-3'-phosphate dehydrogenase (GAPD) in S_ j _ cerevisiae.
  • GAPD glyceraldehyde-3'-phosphate dehydrogenase
  • Plasmid pBGAP is similar in structure to pl25N, described above, with the GAPD promoter being substituted for the PH05 promoter; a somewhat diffferent method, however, was used to construct the two plasmids.
  • the GAPD promoter " is contained within a 2.1 kb Hindlll genomic DNA fragment from S_-_ cerevisiae, which includes the DNA coding sequence for the GAPD gene, as well as the 5' and 3' flanking regions.
  • the 2.1 kb Hindlll fragment is completely digested with Hpal endonuclease to* ield a 1.4 kb and a 0.7 kb fragment.
  • the 1.4 kb fragment is then isolated and digested completely with TaqI; from these digests a 650-bp fragment containing the GAPD promoter region is isolated.
  • BamHI linkers are ligated to both ends with T4 DNA ligase.
  • Subsequent reaction with BamHI and EcoRI produces a 650-bp DNA fragment, which can be isolated from a gel and then inserted into the previously described 6.87 kb vector fragment containing BamHI and EcoRI insertion sites, and ligated to form a circular plasmid.
  • Two kinds of BamHI-EcoRI-ended promoter fragments can be isolated in this way: One with the EcoRI linker at the 5' end of the promoter and the BamHI linker at the 3' end, and another having a 5' BamHI end and a 3' EcoRI end.
  • Plasmid pl25N can be used for the production and secretion of the ⁇ and 3 subunits of hCG in Saccharomyces cerevisiae; transcription is under the control of the promoter sequence of PH05, and the signal sequence is that of immature ⁇ or 3 hCG.
  • cDNA clones coding for and 3 -hCG, including the signal peptide sequence at the amino-terminal end, are obtained as follows. First, RNA is extracted from placental tissue by the following method: Homogenization of the tissue is carried out in a 1:1 mixture of phenol:lOOmM Na-acetate (pH 5.5) containing ImM EDTA, that has been warmed to 60° for 20 min.
  • RNA is precipitated from the final aqueous phase by the addition of 2.5 volumes of ethanol.
  • placental RNA is passed over oligo (dT)-cellulose in 0.5M NaCl buffered with lOmM Tris-HCl, pH 7.5, and washed with the same solution.
  • Poly A+ mRNA is eluted with lOmM
  • Placental cDNA libraries are constructed by reverse transcription of placental mRNA, second strand synthesis, using E ⁇ coli DNA polymerase I (large fragment), treatment with SI nuclease, and homopolymer tailing (dC) with terminal deoxynucleotidyl transferase; all such procedures are by conventional techniques.
  • a 219 bp fragment of a mouse alpha thyroid stimulating hormone (TSH) clone is used as a hybridization probe.
  • This probe has 77% sequence homology with the human clone. It is radioa ⁇ tively labeled by nick translation and hybridized to the cDNA library under conditions that take into account the extent of ho ology. Strongly hybridizing clones are analyzed by restriction .mapping and clones containing the complete coding sequence of alpha.hCG are verified by DNA sequencing.
  • a 579 bp cDNA fragment encoding beta hCG is described in Fiddes et al. (1980) Nature, Vol. 286, pp. 684-687.
  • EcoRI linkers are first ligated to both ends of the cDNA fragments and then cut with EcoRI to generate cohesive ends.
  • the modified cDNA fragment is then inserted into the PH05 expression vector by cleaving the expression vector with EcoRI, combining the ⁇ or -hCG cDNA with the now-linear expression vector sequence, and ligating these DNA fragments together such that the hCG cDNA is inserted in a counterclockwise transcriptional direction adjacent to and downstream-from the PH05 promoter sequence, as shown in Figs. 3A and 3B, respectively.
  • the resulting expression vectors are used to transform host yeast cells using standard techniques, e.g. that described by Beggs, Nature 275: 104-109 (1978).
  • the transformed yeast cells are cultured in a standard culture medium using standard techniques, e.g.. that summarized by Botstein and Davis, "Principles and Practice of Recombinant'DNA Research with Yeast", _in The Molecular Biology of the Yeast Saccharomyces; Metabolism -and Gene. Expression, pp. 607-636, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982.
  • PH05 promoter-containing expression vectors are used to transform a strain of Saccharomyces cerevisiae carrying a mutant trpl gene which causes tryptophan auxotrophy.
  • Cloning vectors PH05- ⁇ and PH05- 3 each carries an expressible yeast TRP1 gene allowing the transformed yeast (Trp+) to grow in the absence of tryptophan (Trp prototrophy) .
  • Trp transformants are isolated by single-colony isolation on synthetic growth medium lacking tryptophan. This medium, SC-TRP, is described in Table 1, below.
  • Transformants are incubated in LP 3Q (SC-TRP) medium, a low-phosphate medium (Table 1) .
  • Cells retaining cloning vector —PH—0—5.- or —PH—05— 3 can grow in this medium, and begin to synthesize ⁇ or 3 -hCG when 5 the intracellular level of inorganic phosphate begins to decrease.
  • Typical levels are 0.2 mg/1 or greater, as determined by radioimmunoassay.
  • Antigenically active 3 -hCG is found in both the culture medium and in the cells.
  • yeast cells are at first removed from the harvested cell suspension by any of several yeast cells.
  • the cell-free fermentation broth is subjected to conventional protein purification procedures designed to isolate pure ⁇ or 3 -hCG.
  • the cells are suspended in a convenient buffer
  • Figs. 4A and 4B The structure of the GAPD- ⁇ hCG and GAPD- 3 hCG cloning vectors is shown in Figs. 4A and 4B, respectively. These vectors are identical to the PH05- ⁇ hCG and PH05- 3 hCG vectors derived from pl25N, with the GAPD promoter replacing the PH05 promoter.

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EP19850904174 1984-07-31 1985-07-31 Hefeklonierungsvektoren. Withdrawn EP0192679A4 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US63636584A 1984-07-31 1984-07-31
US636365 1984-07-31

Publications (2)

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EP0192679A1 EP0192679A1 (de) 1986-09-03
EP0192679A4 true EP0192679A4 (de) 1988-04-26

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EP (1) EP0192679A4 (de)
JP (1) JPS61503002A (de)
DK (1) DK141386D0 (de)
WO (1) WO1986000923A1 (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4923805A (en) * 1983-11-02 1990-05-08 Integrated Genetics, Inc. Fsh
US5013652A (en) * 1986-10-14 1991-05-07 Genex Corporation Composite yeast vectors

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0116201A1 (de) * 1983-01-12 1984-08-22 Chiron Corporation Sekretorische Expression in Eukaryoten

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2125047B (en) * 1982-08-09 1986-02-19 Ciba Geigy Ag Yeast hybrid vectors and their use for the production of polypeptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0116201A1 (de) * 1983-01-12 1984-08-22 Chiron Corporation Sekretorische Expression in Eukaryoten

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 95, no. 13, 28th September 1981, page 234, abstract no. 110370q, Columbus, Ohio, US; J.C. FIDDES et al.: "The gene encoding the common alpha subunit of the four human glycoprotein hormones", & J. MOL. APPL. GENET. 1981, 1(1), 3-18 *
See also references of WO8600923A1 *

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EP0192679A1 (de) 1986-09-03
DK141386A (da) 1986-03-26
DK141386D0 (da) 1986-03-26
WO1986000923A1 (en) 1986-02-13
JPS61503002A (ja) 1986-12-25

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