WO1986000638A1 - Yeast cloning vehicle - Google Patents
Yeast cloning vehicle Download PDFInfo
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- WO1986000638A1 WO1986000638A1 PCT/US1985/001310 US8501310W WO8600638A1 WO 1986000638 A1 WO1986000638 A1 WO 1986000638A1 US 8501310 W US8501310 W US 8501310W WO 8600638 A1 WO8600638 A1 WO 8600638A1
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- WIPO (PCT)
- Prior art keywords
- yeast
- cloning vehicle
- sequence
- signal
- dna sequence
- Prior art date
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 71
- 238000010367 cloning Methods 0.000 title claims abstract description 32
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 52
- 108091029865 Exogenous DNA Proteins 0.000 claims abstract description 14
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 claims abstract description 13
- 229940088597 hormone Drugs 0.000 claims abstract description 11
- 239000005556 hormone Substances 0.000 claims abstract description 11
- 230000035558 fertility Effects 0.000 claims abstract description 7
- 230000035897 transcription Effects 0.000 claims abstract description 6
- 238000013518 transcription Methods 0.000 claims abstract description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 64
- 239000012634 fragment Substances 0.000 claims description 46
- 108090000623 proteins and genes Proteins 0.000 claims description 38
- 102000004169 proteins and genes Human genes 0.000 claims description 28
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 22
- 150000001413 amino acids Chemical class 0.000 claims description 15
- 108020004414 DNA Proteins 0.000 claims description 14
- 230000014509 gene expression Effects 0.000 claims description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 10
- 108010051457 Acid Phosphatase Proteins 0.000 claims description 5
- 108091058545 Secretory proteins Proteins 0.000 claims description 3
- 102000040739 Secretory proteins Human genes 0.000 claims description 3
- 210000000349 chromosome Anatomy 0.000 claims description 3
- 230000001254 nonsecretory effect Effects 0.000 claims description 2
- YAXNATKKPOWVCP-ZLUOBGJFSA-N Ala-Asn-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O YAXNATKKPOWVCP-ZLUOBGJFSA-N 0.000 claims 1
- 108091081024 Start codon Proteins 0.000 claims 1
- 210000004899 c-terminal region Anatomy 0.000 claims 1
- 239000013612 plasmid Substances 0.000 description 57
- 108091026890 Coding region Proteins 0.000 description 13
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 238000010276 construction Methods 0.000 description 9
- 230000004927 fusion Effects 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 101150012394 PHO5 gene Proteins 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 230000028327 secretion Effects 0.000 description 6
- 108020004635 Complementary DNA Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 5
- 102000014150 Interferons Human genes 0.000 description 5
- 108010050904 Interferons Proteins 0.000 description 5
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 5
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 5
- 238000010804 cDNA synthesis Methods 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 229940079322 interferon Drugs 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000003248 secreting effect Effects 0.000 description 5
- 230000004543 DNA replication Effects 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 125000006850 spacer group Chemical group 0.000 description 4
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 3
- 102100030310 5,6-dihydroxyindole-2-carboxylic acid oxidase Human genes 0.000 description 3
- 101710163881 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 description 3
- 102000013563 Acid Phosphatase Human genes 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 2
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 108020005091 Replication Origin Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000014621 translational initiation Effects 0.000 description 2
- 101150108727 trpl gene Proteins 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 108010058643 Fungal Proteins Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 101710149086 Nuclease S1 Proteins 0.000 description 1
- 101710093417 Phosphate-repressible acid phosphatase Proteins 0.000 description 1
- 101100097319 Schizosaccharomyces pombe (strain 972 / ATCC 24843) ala1 gene Proteins 0.000 description 1
- 101150006914 TRP1 gene Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 230000001036 exonucleolytic effect Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000001279 glycosylating effect Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000037041 intracellular level Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000005257 nucleotidylation Effects 0.000 description 1
- 230000007030 peptide scission Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000000063 preceeding effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
Classifications
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- 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]
-
- 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
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- 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
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
Definitions
- This invention relates to cloning vehicles for production of proteins.
- proteins is used in this application to include peptides of indefinite size.
- proteins exported from eukaryotic cells are processed in a secretory pathway that involves synthesis of a precursor protein (containing an amino-teri ⁇ inal "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.
- a precursor protein containing an amino-teri ⁇ inal "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 Schekman and Novick, "The secretory process and yeast cell-surface assembly", in The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, Strathern et al. (Eds.), Cold Spring Harbor Laboratory, 1982]. Moreover, unlike an analogous secretory pathway that exists in some prokaryotic species, the 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 interferon signal sequence. The mature secreted protein reported by Hitzeman et al. revealed 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 PHO5 gene of the yeast Saccharomyces cerevisiae (containing the promoter and 80% of the 5' end of the PHO5 signal sequence) to a cDNA fragment coding for the mature protein sequence and a portion of the 3' end of the signal sequence of human alpha interferon.
- PHO5 codes for the major repressible (by phosphate) acid phosphatase, a secreted enzyme in this yeast.
- 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 generally a cloning vehicle that is used to transform a host cell, preferably a yeast cell, for expression and secretion of a protein by the cell.
- the cloning vehicle includes in order of transcription: a yeast transcription control DNA sequence, a DNA sequence coding for a complete yeast signal peptide, and a DNA sequence coding for a human fertility hormone or a sub-unit thereof.
- the hormone is the alpha sub-unit of human chorionic gonadotropin (alpha HCG), and the yeast signal sequence is identical to the naturally occurring yeast PHO5 signal peptide.
- the above described cloning vehicle enables expression of a human fertility hormone or a sub-unit thereof in yeast, so that the hormone is secreted by the yeast into the surrounding medium. Moreover, the protein is recovered in its mature form after processing (i.e., removal of the yeast signal sequence). Control over the placement and spacing of the signal sequence, as described below, will enable the desired fertility hormone to be expressed and processed by the host organism, such that cleavage of the signal sequence will occur at the correct site and the mature form of the hormone will be secreted.
- the information necessary to effect the correct processing of the mature protein is contained in the yeast signal DNA sequence, and is not dependent on the nature of the DNA sequence coding for the exogenous protein. Description of the Preferred Embodiment
- Figure 1 is a diagram representing plasmid 99NMlu-alpha.
- Figure 2 is a diagram representing plasmid 99N.
- Figure 3 is a diagram representing plasmid
- Figure 4 is a diagram representing plasmid 99N-alpha.
- Figure 5 is a partial restriction map of a DNA fragment cloned into pBR322, including the coding sequence for the first 30 nucleotides of mature alpha
- the components of the cloning vector or vehicle are illustrated by plasmid 99NMlu-alpha, depicted in Fig. 1, and, alternatively, by p99N-alpha in Fig. 4.
- the plasmid includes a yeast transcription control sequence which comprises a functional promoter region and a transcription initiation site.
- the functional yeast promoter region should include sufficient sequences upstream from other plasmid components to permit initiation of transcription, for example, a complete naturally occurring yeast promoter. (Naturally occurring yeast transcription control DNA sequences containing certain deletions at the 3' end are also functional promoters.)
- the 5' to 3' DNA sequence from the BamHI site (-553) to the nucleotide preceding the ATG triplet (defined as +1, +2, +3), represents a functional PHO5 promoter region with sufficient upstream sequences to permit initiaton of transcription. Certain deletions of the 3' end of the PH05 transcription control region are also functional promoters.
- Fused in phase with the transcription control sequence is a sequence which is transcribed and translated into the signal peptide of a yeast secretory protein.
- p99NMlu-alpha codes for a signal peptide (identical to the PHO5 signal peptide), having the following amino acid sequence: Met-Phe-Lys-Ser-Val-ValTyr-Ser-Ile-Leu-Ala-Ala-Ser-Leu-Ala-Asn-Ala.
- the DNA sequence coding for the signal peptide on p99NMlu-alpha is: ATGTTTAAATCTGTTGTTTATTCAATTTTAGCCGCTTCTTTGGCCAACGCG.
- Exogenous protein means a protein that, in naturally occurring systems, is not produced under control of the yeast promoter of the vehicle, and is not produced with the yeast signal peptide of the vehicle. This term includes yeast proteins, but preferably the exogenous DNA sequence codes for the mature form of a nonyeast protein.
- Changes can be made in naturally occurring signal DNA sequences to facilitate fusion of the signal sequence to the sequence coding for the mature protein, but one should avoid changes which are reflected in the amino acid sequence of the resulting signal peptide.
- the 3' end of the signal DNA sequence is ligated to the 5' end of the exogenous DNA sequence such that there are no extraneous nucleotides between the sequences. In this way, the nascent protein will contain a signal peptide directly adjacent to the desired mature protein and the protein that is secreted should not contain extraneous amino acids.
- plasmid p99N DNA replication origins and DNA fragments conferring phenotypic bases for plasmid selection.
- the detailed features which are present in p99NMlu-alpha are illustrated by the following description of its precursor, plasmid p99N, and of the method of making p99NMlu-alpha from p99N.
- the above-described cloning vehicle can be constructed by engineering a vector containing a yeast transcription control sequence and a yeast signal sequence which has been modified to provide a multipurpose cloning site.
- Plasmid 99NMlu (Fig. 3) is such a vector containing a cloning site Mlul.
- the restriction site should be positioned at or near the end of the yeast signal DNA sequence, so that any exogenous DNA sequence coding for a desired secretory or nonsecretory protein, which has been engineered by restriction digest or by the use of synthetic linkers, is then inserted into the Mlul site of plasmid 99NMlu, and expression and secretion of the desired protein or peptide is achieved.
- the linker used may be engineered to maintain the intact naturally occurring signal DNA sequence, but the redundancy of the genetic code provides some flexibility as to the engineering steps performed. Similarly, engineering and nucleotide additions may be required at the 5' end of the exogenous DNA sequence to insure an inphase fusion to the signal sequence.
- An example of the structure and construction of such linkers is outlined below for p99NMlu-alpha.
- Plasmid 99N includes the following DNA segments (counter-clockwise from the EcoRl site):
- the functional TRP1 gene from yeast contained within a 1.45 kb EcoRI genomic DNA fragment from chromosome 4, described in Kingsman et al. (1979) Gene 7 , 141-152 and in Tschumper and Carbon (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 and Carbon (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.
- HincI I fragment contains a "strong" origin of DNA replication, (i.e., “stronger” than ars1 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 99N is not important for this function;
- Plasmid 99N is constructed as follows. Plasmid
- YRp7 is partially digested with EcoRI such that only one of the two EcoRI sites is cleaved. Most of the 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).
- 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 to the TRP1 promoter, is identified by restriction mapping.
- the 1.45 kb HincI I fragment from the yeast 2 micron plasmid is ligated into the Nrul site of YRp7'.
- the plasmid that is generated,. YRp7'N is digested with both EcoRI and BamHI. The large fragment.
- the PHQ5 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 77, 6541-6545.
- the PH05 fragment is prepared by digesting a plasmid carrying the 8 kb EcoRI PH05 region (e.g. pAP20, as described in Anderson, Thill and Kramer. Molec. Cell Biol.
- the composition of the Kpnl-EcoRI spacer fragment is arbitrary.
- the spacer fragment consists of PH05 structural sequences beginning at the Kpnl site (at +94bp) and terminating at the Pstl site (at +1500 bp) to which had been added an EcoRI linker sequence.
- the fragment is combined with the PH05 fragment and the vector fragment into a circular plasmid with DNA ligase. This product is used to transform E. coli to ampicillin resistance. From this pool of transformants plasmid 99N is identified and plasmid DNA is prepared. Plasmid 99N has been deposited with the NRRL and bears accession number 15790.
- Plasmid 99N can be used to construct a cloning vehicle (for example p99Nalpha) for an exogenous DNA sequence beginning with a G as the first base pair using the technique described below in the section "Plasmid 99N-alpha". Alternatively, the insertion of a Mlul site in p99N would allow expression, of exogenous DNA without regard to the identity of the initial base pair. Plasmid 99NMlu.
- the following engineering steps are required. First, the PH05 promoter and signal sequence containing 1.95kb BamHI-EcoRI fragment of 99N is inserted into pBR322 to generate plasmid pBRPho. Then, the Ball site in the pBR portion of pBRPho is eliminated by removing the
- pAPP now has only one Ball site which is located close to the 3' end of the DNA sequence encoding the PH05 signal.
- the next step involves the replacement of the Kpnl site located next to the Ball site with an Mlul site.
- pAPP is digested with Kpnl and the 3' overhang is removed using T4 DNA polymerase.
- Synthetic Mlul linkers (ACGCGT) are ligated on followed by digestion with Mlul and ligation to close the vector. Bacterial transformants are checked for the presence of a Mlul site; such a plasmid.
- pAPPM is isolated.
- pAPPM is digested with Mlul and synthetic 7-mers (5'TGGCCAA3') and 11-mers (5'CGCGTTGGCCA3') that contain a Ball site and Mlul overhang are ligated on.
- Several of these linkers can be ligated in-between the two Mlul overhangs which leads to the following structure: (PH05 Signal-Bal... Mlu-Bal........Bal-Mlu).
- alpha hCG human chorionic gonadotropin
- Saccharomyces cerevisiae directed by the promoter, translation initiation site and signal sequence of PH05.
- alpha hCG and yeast acid phosphatase are secretory proteins derived from pre-proteins containing a signal peptide at their amino terminal end.
- the first 50 nucleotides of the coding sequence of the mature alpha hCG contain no recognition sequences for any known restriction enzymes. A fusion of the mature portion of alpha hCG to any signal sequence would thus require the extensive use of very long synthetic DNA linkers.
- This can be circumvented by creation of a restriction site downstream from the start of the mature coding sequence at positions that contain all but one or two nucleotides of a restriction enzyme recognition site. Relatively short and inexpensive synthetic DNA fragments can then be inserted between the newly created restriction site and the Mlul site of 99NMlu.
- the DNA fragment coding for the mature form of alpha hCG can be engineered so that a correct in-phase fusion to the PH05 signal in plasmid 99NMlu can be achieved.
- Exonuclease Ba131 can be used to resect this fragment from the BamHI site under conditions in which about 90 bp will be digested.
- EcoRI linkers which contain a C at their 3' end can be ligated. Only molecules in which the resection had ended exactly at the sequence 5'TGCAG3' will - after addition of a C - contain a Pstl site at postion 94.
- the above mentioned ligation mixture can be cut with Pstl and fragments of about 250bp length which contain a Pstl site at each end can be selected by cloning into the Pstl site of pBR322.
- Plasmids containing such a f ragment can be diges ted with Ps tl and synthetic 10-mers (5'CATCAGGAGC3') and 18-mers (5'CGCGGCTCCTGATGTGCA3') are ligated on. These linkers contain a Mlul overhang and the missing alpha hCG coding sequence ending with a Pstl overhang. This ligation mixture can then be digested with Mlul and the fragment can be cloned into the Mlul site of pPhoM.
- the 3' fragment of the alpha hCG coding region can be inserted into this plasmid as a Xbal-EcoRI fragment and the entire BamHI-EcoRI fragment containing the PH05 promoter and the PH05 signal fused to the mature alpha hCG coding region can be inserted into p99NMlu to yield p99NMlualpha which then can be used to transform yeast. Plasmid 99N-alpha.
- plasmid 99N was used as a vector for constructing a hybrid gene in which the PH05 signal sequence was fused to the DNA sequence coding for the mature form of alpha hCG to yield plasmid 99N-alpha, illustrated in Fig. 4.
- the specific construction of the hybrid gene was possible because the PH05 signal sequence was manipulated to contain only the first 51 nucleotides. coding for the entire 17 amino acid signal peptide, plus an additional guanine (G) residue. This was achieved by first digesting the PH05 DNA with Kpnl enzyme to generate a 3'protruding strand. Then, flush ended molecules were obtained by the 3'-5'exonucleolytic activity of T4 DNA polymerase.
- the coding region of the mature alpha hCG was manipulated in a similar way as described above for making p99NMlu-alpha. Addition of a GA to the mature coding region creates a new restriction site SacI with the recognition sequence GAGCTC. This has been achieved by ligating alpha-hCG fragments which had been resected with Bal31 from the BamHI site to fragments that contained a filled in Sall site and therefore a GA at their ends. The newly created SacI site contains within it an Alul site (AGCT). Cutting with Alul yields fragments which have the first G of the first amino acid of the mature alpha-hCG missing.
- AGCT Alul site
- Plasmid 99N-alpha has been deposited with the NRRL and bears accession number B 15791. Protein Synthesis
- the cloning vehicle is used to transform a host yeast organism using standard techniques such as described by Beggs, Nature 275:104-109 (1978).
- the transformed yeast organism is cultured in a standard culture medium using standard techniques such as those 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.
- the secreted mature polypeptide is recovered from the surrounding medium.
- Plasmid 99N-alpha was used to transform a strain of Saccharomyces cerevisiae carrying a mutant trpl gene (causing tryptophan auxotrophy) to the Trp+ phenotype (tryptophan prototrophy). Plasmid 99N-alpha carries an expressible yeast trpl gene allowing the transformed yeast to grow in the absence of tryptophan (Trp prototrophy). Suitable methods for the transformation of yeast by plasmid DNA are described for example by Beggs, 1978. One or more Trp + transformants were isolated by single-colony isolation on synthetic growth medium lacking tryptophan. This 5 medium is called SC-TRP and is described in Table 1.
- the transformed strain was incubated in a low-phosphate synthetic liquid growth medium that lacks tryptophan and contains 30 mg/l KH 2 PO 4 and 1.5 g/l KC1.
- This medium is called LP 30 (SC—TRP) and is described in Table 1.
- Cells retaining the expression of plasmid 99N-alpha grow in this medium and begin to synthesize alpha hCG when the intracellular level of inorganic phosphate begins to decrease. After two days of incubation at 30 degrees C, virtually all (greater than 90%) of the antigenicall ctive alpha hCG was found in the culture medium. Typical levels were 0.2 mg/l or greater, as determined by radioimmunoassay.
- the yeast cells were at first removed from the harvested cell suspension by any of several convenient means such as centrifugation, filtration, etc. Then, the cell-free fermentation broth was subjected to appropriate protein purification procedures designed to isolate pure alpha hCG.
- plasmid 99NMlu can be used to attach the PH05 signal sequence to any foreign gene or cDNA and can be introduced to any strain of Saccharomyces cerevisiae.
- the transformed yeast can then be used to produce the desired foreign protein using techniques such as those described above for production of alpha hCG using yeast transformed with p99N alpha.
- the system provides an increased yield of the foreign protein which is secreted into the extra cellular fluid.
- the three terminal amino acids should be identical to the naturally occurring yeast signal sequence amino acids. While it is desirable to produce an intact, unmodified signal peptide, one skilled in the art may be able to use the claimed invention while making a few such changes. Similarly, one may include a few extraneous amino acids at the N-terminal end of the desired polypeptide and tolerate some slight (3-6 base pairs) movement of the restriction enzyme site with respect to the end of the signal DNA sequence.
- fertility hormones that can be expressed and secreted include: lactizing Hormone Follicle Stimulating.
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Abstract
A cloning vehicle with a yeast transcription control sequence and a yeast signal sequence placed in correct reading frame and transcription direction with respect to an exogenous DNA sequence coding for a human fertility hormone or a sub-unit thereof.
Description
Yeast Cloning Vehicle
BACKGROUND OF THE INVENTION
This invention relates to cloning vehicles for production of proteins. (The term "proteins" is used in this application to include peptides of indefinite size.)
There has been increasing interest in the use of eukaryotic cells, such as yeasts, as hosts for the expression of genes or complementary DNA (cDNA) coding sequences for specific proteins. Typically, proteins exported from eukaryotic cells are processed in a secretory pathway that involves synthesis of a precursor protein (containing an amino-teriαinal "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 Schekman and Novick, "The secretory process and yeast cell-surface assembly", in The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, Strathern et al. (Eds.), Cold Spring Harbor Laboratory, 1982]. Moreover, unlike an analogous secretory pathway that exists in some prokaryotic species, the 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 interferon signal sequence. The mature secreted protein reported by Hitzeman et al. revealed 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 PHO5 gene of the yeast
Saccharomyces cerevisiae (containing the promoter and 80% of the 5' end of the PHO5 signal sequence) to a cDNA fragment coding for the mature protein sequence and a portion of the 3' end of the signal sequence of human alpha interferon. PHO5 codes for the major repressible (by phosphate) acid phosphatase, a secreted enzyme in this yeast. The Hinnen et al. construction reportedly lacked the DNA coding sequence for the last 3 amino acids of the PHO5 signal peptide and instead the last three amino acids of the hybrid signal were 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.
Summary of the Invention In a first aspect, the invention features generally a cloning vehicle that is used to transform a host cell, preferably a yeast cell, for expression and secretion of a protein by the cell. The cloning vehicle includes in order of transcription: a yeast transcription control DNA sequence, a DNA sequence coding for a complete yeast signal peptide, and a DNA sequence coding for a human fertility hormone or a sub-unit thereof.
In preferred embodiments, the hormone is the alpha sub-unit of human chorionic gonadotropin (alpha HCG), and the yeast signal sequence is identical to the naturally occurring yeast PHO5 signal peptide.
The above described cloning vehicle enables expression of a human fertility hormone or a sub-unit
thereof in yeast, so that the hormone is secreted by the yeast into the surrounding medium. Moreover, the protein is recovered in its mature form after processing (i.e., removal of the yeast signal sequence). Control over the placement and spacing of the signal sequence, as described below, will enable the desired fertility hormone to be expressed and processed by the host organism, such that cleavage of the signal sequence will occur at the correct site and the mature form of the hormone will be secreted. The information necessary to effect the correct processing of the mature protein is contained in the yeast signal DNA sequence, and is not dependent on the nature of the DNA sequence coding for the exogenous protein. Description of the Preferred Embodiment
Drawings
Figure 1 is a diagram representing plasmid 99NMlu-alpha.
Figure 2 is a diagram representing plasmid 99N. Figure 3 is a diagram representing plasmid
99NMlu.
Figure 4 is a diagram representing plasmid 99N-alpha.
Figure 5 is a partial restriction map of a DNA fragment cloned into pBR322, including the coding sequence for the first 30 nucleotides of mature alpha
HCG.
Cloning Vehicle Structure
The components of the cloning vector or vehicle are illustrated by plasmid 99NMlu-alpha, depicted in Fig. 1, and, alternatively, by p99N-alpha in Fig. 4.
The plasmid includes a yeast transcription control sequence which comprises a functional promoter region and a transcription initiation site.
The functional yeast promoter region should include sufficient sequences upstream from other plasmid components to permit initiation of transcription, for example, a complete naturally occurring yeast promoter. (Naturally occurring yeast transcription control DNA sequences containing certain deletions at the 3' end are also functional promoters.) The 5' to 3' DNA sequence from the BamHI site (-553) to the nucleotide preceding the ATG triplet (defined as +1, +2, +3), represents a functional PHO5 promoter region with sufficient upstream sequences to permit initiaton of transcription. Certain deletions of the 3' end of the PH05 transcription control region are also functional promoters.
Fused in phase with the transcription control sequence is a sequence which is transcribed and translated into the signal peptide of a yeast secretory protein. p99NMlu-alpha codes for a signal peptide (identical to the PHO5 signal peptide), having the following amino acid sequence: Met-Phe-Lys-Ser-Val-ValTyr-Ser-Ile-Leu-Ala-Ala-Ser-Leu-Ala-Asn-Ala. The DNA sequence coding for the signal peptide on p99NMlu-alpha is: ATGTTTAAATCTGTTGTTTATTCAATTTTAGCCGCTTCTTTGGCCAACGCG. The above yeast DNA sequences are positioned in phase with the DNA sequences coding for the desired exogenous protein. "Exogenous protein" means a protein that, in naturally occurring systems, is not produced under control of the yeast promoter of the vehicle, and is not produced with the yeast signal peptide of the vehicle. This term includes yeast proteins, but
preferably the exogenous DNA sequence codes for the mature form of a nonyeast protein.
Changes can be made in naturally occurring signal DNA sequences to facilitate fusion of the signal sequence to the sequence coding for the mature protein, but one should avoid changes which are reflected in the amino acid sequence of the resulting signal peptide. The 3' end of the signal DNA sequence is ligated to the 5' end of the exogenous DNA sequence such that there are no extraneous nucleotides between the sequences. In this way, the nascent protein will contain a signal peptide directly adjacent to the desired mature protein and the protein that is secreted should not contain extraneous amino acids.
Other components of the preferred cloning vehicle include DNA replication origins and DNA fragments conferring phenotypic bases for plasmid selection. The detailed features which are present in p99NMlu-alpha are illustrated by the following description of its precursor, plasmid p99N, and of the method of making p99NMlu-alpha from p99N. Cloning Vehicle Construction and Precursors
The above-described cloning vehicle can be constructed by engineering a vector containing a yeast transcription control sequence and a yeast signal sequence which has been modified to provide a multipurpose cloning site. Plasmid 99NMlu (Fig. 3) is such a vector containing a cloning site Mlul. The restriction site should be positioned at or near the end of the yeast signal DNA sequence, so that any exogenous DNA sequence coding for a desired secretory or nonsecretory protein, which has been engineered by
restriction digest or by the use of synthetic linkers, is then inserted into the Mlul site of plasmid 99NMlu, and expression and secretion of the desired protein or peptide is achieved. Depending on where the restriction site is positioned and what sequences are required, the linker used may be engineered to maintain the intact naturally occurring signal DNA sequence, but the redundancy of the genetic code provides some flexibility as to the engineering steps performed. Similarly, engineering and nucleotide additions may be required at the 5' end of the exogenous DNA sequence to insure an inphase fusion to the signal sequence. An example of the structure and construction of such linkers is outlined below for p99NMlu-alpha.
What follows is a specific description of p99N and its construction, followed by a description of the construction of p99NMlu from p99N by inserting the restriction site Mlul. Exogenous DNA is then spliced into p99NMlu to create p99NMlu-alpha. Routine recombinant DNA procedures that may be used in this construction are described in Maniatis et al. Molecular Cloning: A Laboratory Manual, Coldspring Harbor Laboratory, Cold Spring Harbor, 1982. Plasmid 99N. Plasmid 99N includes the following DNA segments (counter-clockwise from the EcoRl site):
1. The functional TRP1 gene from yeast contained within a 1.45 kb EcoRI genomic DNA fragment from chromosome 4, described in Kingsman et al. (1979) Gene 7 , 141-152 and in Tschumper and Carbon (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 and Carbon (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 pBR322 sequence, EcoRI-NruI, 3389 bp, carrying the gene for ampicillin resistance in E. coli. as well as a DNA replication origin that functions in E. coli;
3. The 1.45 kb HincI I 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 Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, 1981. The HincI I fragment contains a "strong" origin of DNA replication, (i.e., "stronger" than ars1 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 99N is not important for this function;
4. The pBR322 5.97 bp sequence from Nrul-BamHI;
5. The 0.6 kb BamHI-Kpnl segment of the 8 kb ECoRI-PH05 fragment. This fragment contains the intact PH05 promoter plus all of the DNA sequence necessary to encode the 17 amino acid signal peptide (including the initial methionine); and
6. The Kpnl-EcoRI fragment is used as a spacer region. The source and length of this fragment is not important for its use in plasmid 99N or its derivatives. In plasmid 99N, the spacer region consists of PH05 structural sequences. Plasmid 99N is constructed as follows. Plasmid
YRp7 is partially digested with EcoRI such that only one of the two EcoRI sites is cleaved. Most of the 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). (Alternatively, 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 to the TRP1 promoter, is identified by restriction mapping. Next, the 1.45 kb HincI I fragment from the yeast 2 micron plasmid is ligated into the Nrul site of YRp7'. The plasmid that is generated,. YRp7'N, is digested with both EcoRI and BamHI. The large fragment. 6.87 kb, which is isolated from the gel, is the vector fragment.
The PHQ5 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 77, 6541-6545. The PH05 fragment is prepared by digesting a plasmid carrying the 8 kb EcoRI PH05 region (e.g. pAP20, as described in Anderson, Thill and Kramer. Molec. Cell Biol. 3:562-569, 1983). with BamHI and Kpnl and isolating the 0.6 kb BamHI-Kpnl fragment from a gel. The 5' to 3' DNA sequence from the BamHI site (-553) to the nucleotide preceeding the ATG triplet represents a. functional PH05 promoter region with sufficient upstream sequences to permit initiation of transcription.
The composition of the Kpnl-EcoRI spacer fragment is arbitrary. In plasmid 99N, the spacer fragment consists of PH05 structural sequences beginning at the Kpnl site (at +94bp) and terminating at the Pstl site (at +1500 bp) to which had been added an EcoRI linker sequence. The fragment is combined with the PH05 fragment and the vector fragment into a circular plasmid with DNA ligase. This product is used to transform E. coli to ampicillin resistance. From this pool of transformants plasmid 99N is identified and plasmid DNA is prepared. Plasmid 99N has been deposited with the NRRL and bears accession number 15790.
Plasmid 99N can be used to construct a cloning vehicle (for example p99Nalpha) for an exogenous DNA sequence beginning with a G as the first base pair using the technique described below in the section "Plasmid
99N-alpha". Alternatively, the insertion of a Mlul site in p99N would allow expression, of exogenous DNA without regard to the identity of the initial base pair. Plasmid 99NMlu.
In order to place the Mlul site within the 3' end of the PH05 signal sequence of 99N, the following engineering steps are required. First, the PH05 promoter and signal sequence containing 1.95kb BamHI-EcoRI fragment of 99N is inserted into pBR322 to generate plasmid pBRPho. Then, the Ball site in the pBR portion of pBRPho is eliminated by removing the
Aval-PvuII fragment to generate pAPP. pAPP now has only one Ball site which is located close to the 3' end of the DNA sequence encoding the PH05 signal.
The next step involves the replacement of the Kpnl site located next to the Ball site with an Mlul site. In order to accomplish this replacement pAPP is digested with Kpnl and the 3' overhang is removed using T4 DNA polymerase. Synthetic Mlul linkers (ACGCGT) are ligated on followed by digestion with Mlul and ligation to close the vector. Bacterial transformants are checked for the presence of a Mlul site; such a plasmid. pAPPM, is isolated. pAPPM is digested with Mlul and synthetic 7-mers (5'TGGCCAA3') and 11-mers (5'CGCGTTGGCCA3') that contain a Ball site and Mlul overhang are ligated on. Several of these linkers can be ligated in-between the two Mlul overhangs which leads to the following structure: (PH05 Signal-Bal... Mlu-Bal........Bal-Mlu).
These structures are digested with Ball and the vector is closed by ligation. This removes all the unnecessary
linkers and moves the Mlul site next to the Ball site in the PH05 signal, thus giving the desired configuration, which is verified by DNA sequencing (pPhoM).
Construction of 99NMlu from plasmids 99N and pPhoM then is accomplished as follows: The 1.95kb BamHI-EcoRI fragment of pPhoM is isolated and ligated into 99N which has been cut with both BamHI and EcoRI and treated with bacterial acid phosphatase. Bacterial transformants are screened for plasmids having an Mlul site. Such a plasmid, 99NMlu, described in Figure 3, is isolated and can be used for fusions of the PH05 signal to foreign proteins. Plasmid 99NMlu has been deposited with the NRRL and bears accession number B 15792.
Thus, when 99NMlu is digested with Mlul. a 4-base overhang is created which ends exactly at the site encoding the carboxyl terminal of the signal sequence. This Mlul overhang can be modified so that either the site is filled in with dNTP's using the large fragment of DNA polymerase I (Klenow enzyme) which results in a blunt ended complete PH05 signal sequence or alternatively, the overhang is digested with nuclease S1 which leads to a blunt ended PH05 signal sequence of which the last 4 nucleotides are missing. This region of 99NMlu can also be cut with FnuDII (recognition sequence CGCG) which yields a blunt ended PH05 signal of which the last 2 nucleotides are missing.
These modifications allow fusion of the PH05 signal in frame to any coding region that is engineered by restriction digest or use of synthetic linkers:
Plasmid 99NMlu alpha.
The following example describes the proposed synthesis and secretion of the alpha subunit of human chorionic gonadotropin (alpha hCG) in Saccharomyces cerevisiae directed by the promoter, translation initiation site and signal sequence of PH05. Both alpha hCG and yeast acid phosphatase are secretory proteins derived from pre-proteins containing a signal peptide at their amino terminal end.
The first 50 nucleotides of the coding sequence of the mature alpha hCG contain no recognition sequences for any known restriction enzymes. A fusion of the mature portion of alpha hCG to any signal sequence would thus require the extensive use of very long synthetic DNA linkers. This can be circumvented by creation of a restriction site downstream from the start of the mature
coding sequence at positions that contain all but one or two nucleotides of a restriction enzyme recognition site. Relatively short and inexpensive synthetic DNA fragments can then be inserted between the newly created restriction site and the Mlul site of 99NMlu. In a preferred method, the DNA fragment coding for the mature form of alpha hCG can be engineered so that a correct in-phase fusion to the PH05 signal in plasmid 99NMlu can be achieved.
An alpha hCG clone coding for the complete pre-alpha hCG molecule had been cloned between the BamHI and EcoRI sites of pBR322. A partial restriction map of this insert and the sequence of the first 30 nucleotides of the coding sequence for the mature alpha hCG are shown in Fig. 5. The codon for the first amino acid of the mature alpha hCG starts at the eighty-fifth base as indicated by the arrow. Replacement of the G at position 94 with a C would create a Pstl site (CTGCAG) which then could be used to insert synthetic DNA linkers. Exonuclease Ba131 can be used to resect this fragment from the BamHI site under conditions in which about 90 bp will be digested. To these digested fragments, EcoRI linkers which contain a C at their 3' end can be ligated. Only molecules in which the resection had ended exactly at the sequence 5'TGCAG3' will - after addition of a C - contain a Pstl site at postion 94. Thus, the above mentioned ligation mixture can be cut with Pstl and fragments of about 250bp length which contain a Pstl site at each end can be selected by cloning into the Pstl site of pBR322. Plasmids containing such a f ragment can be diges ted with Ps tl and synthetic 10-mers (5'CATCAGGAGC3') and 18-mers
(5'CGCGGCTCCTGATGTGCA3') are ligated on. These linkers contain a Mlul overhang and the missing alpha hCG coding sequence ending with a Pstl overhang. This ligation mixture can then be digested with Mlul and the fragment can be cloned into the Mlul site of pPhoM. The 3' fragment of the alpha hCG coding region can be inserted into this plasmid as a Xbal-EcoRI fragment and the entire BamHI-EcoRI fragment containing the PH05 promoter and the PH05 signal fused to the mature alpha hCG coding region can be inserted into p99NMlu to yield p99NMlualpha which then can be used to transform yeast. Plasmid 99N-alpha.
In an alternative method, plasmid 99N was used as a vector for constructing a hybrid gene in which the PH05 signal sequence was fused to the DNA sequence coding for the mature form of alpha hCG to yield plasmid 99N-alpha, illustrated in Fig. 4. The specific construction of the hybrid gene was possible because the PH05 signal sequence was manipulated to contain only the first 51 nucleotides. coding for the entire 17 amino acid signal peptide, plus an additional guanine (G) residue. This was achieved by first digesting the PH05 DNA with Kpnl enzyme to generate a 3'protruding strand. Then, flush ended molecules were obtained by the 3'-5'exonucleolytic activity of T4 DNA polymerase. The coding region of the mature alpha hCG was manipulated in a similar way as described above for making p99NMlu-alpha. Addition of a GA to the mature coding region creates a new restriction site SacI with the recognition sequence GAGCTC. This has been achieved by ligating alpha-hCG fragments which had been resected
with Bal31 from the BamHI site to fragments that contained a filled in Sall site and therefore a GA at their ends. The newly created SacI site contains within it an Alul site (AGCT). Cutting with Alul yields fragments which have the first G of the first amino acid of the mature alpha-hCG missing. The fusion of those fragments to the modified signal peptide in 99N resulted in the construction of plasmid 99N-alpha which contains an intact PH05 promoter, translation initiation site and complete signal sequence fused in phase to the sequence coding for mature alpha hCG.
Plasmid 99N-alpha has been deposited with the NRRL and bears accession number B 15791. Protein Synthesis
The cloning vehicle is used to transform a host yeast organism using standard techniques such as described by Beggs, Nature 275:104-109 (1978).
The transformed yeast organism is cultured in a standard culture medium using standard techniques such as those 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. The secreted mature polypeptide is recovered from the surrounding medium.
Plasmid 99N-alpha was used to transform a strain of Saccharomyces cerevisiae carrying a mutant trpl gene (causing tryptophan auxotrophy) to the Trp+ phenotype (tryptophan prototrophy). Plasmid 99N-alpha carries an expressible yeast trpl gene allowing the transformed yeast to grow in the absence of tryptophan
(Trp prototrophy). Suitable methods for the transformation of yeast by plasmid DNA are described for example by Beggs, 1978. One or more Trp+ transformants were isolated by single-colony isolation on synthetic growth medium lacking tryptophan. This 5 medium is called SC-TRP and is described in Table 1.
The transformed strain was incubated in a low-phosphate synthetic liquid growth medium that lacks tryptophan and contains 30 mg/l KH2PO4 and 1.5 g/l KC1. This medium is called LP30 (SC—TRP) and is described in Table 1. Cells retaining the expression of plasmid 99N-alpha grow in this medium and begin to synthesize alpha hCG when the intracellular level of inorganic phosphate begins to decrease. After two days of incubation at 30 degrees C, virtually all (greater than 90%) of the antigenicall ctive alpha hCG was found in the culture medium. Typical levels were 0.2 mg/l or greater, as determined by radioimmunoassay.
Since most or all of the alpha hCG was secreted into the culture medium, the yeast cells were at first removed from the harvested cell suspension by any of several convenient means such as centrifugation, filtration, etc. Then, the cell-free fermentation broth was subjected to appropriate protein purification procedures designed to isolate pure alpha hCG.
As described previously, plasmid 99NMlu can be used to attach the PH05 signal sequence to any foreign gene or cDNA and can be introduced to any strain of Saccharomyces cerevisiae. The transformed yeast can then be used to produce the desired foreign protein using techniques such as those described above for production of alpha hCG using yeast transformed with p99N alpha. The system provides an increased yield of the foreign protein which is secreted into the extra cellular fluid.
Each of the plasmid deposits referred to in this application has been made under conditions which: (a) provide for access to the culture during pendency of the patent application to one determined by the
Commissioner of the U.S. Patent and Tredemark Office to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122; and (b) ensure that all restrictions on the availability to the public of the culture so deposited will be irrevocably removed upon the granting of the patent. Other Embodiments
Other embodiments are within the following claims. To achieve adequate signal cleavage site recognition in the expressed polypeptide, the three terminal amino acids should be identical to the naturally occurring yeast signal sequence amino acids. While it is desirable to produce an intact, unmodified signal peptide, one skilled in the art may be able to use the claimed invention while making a few such changes. Similarly, one may include a few extraneous amino acids at the N-terminal end of the desired polypeptide and tolerate some slight (3-6 base pairs) movement of the restriction enzyme site with respect to the end of the signal DNA sequence.
Other fertility hormones that can be expressed and secreted include: Leuteinizing Hormone Follicle Stimulating.
Claims
1. A cloning vehicle capable of effecting the expression of an exogenous DNA sequence in a yeast host, said cloning vehicle comprising, in phase and in order of transcription: a yeast transcription-control sequence, a yeast signal sequence which codes for a yeast signal peptide, and an exogenous DNA sequence which comprises a DNA sequence coding for a human fertility hormone or a sub-unit thereof.
2. The cloning vehicle of claim 1 wherein said yeast signal sequence and said exogenous DNA sequence code for a protein comprising an intact yeast signal peptide abutting the desired human fertility hormone with no extraneous amino acids therebetween.
3. The cloning vehicle of claim 1 wherein said yeast transcription control sequence is substantially identical to the naturally occurring transcription-control sequence of a phosphaterepressible yeast acid phosphatase gene.
4. The cloning vehicle of claim 3 wherein said yeast transcription control DNA sequence is substantially identical to the transcription control sequence of a yeast PH05 gene.
5. The cloning vehicle of claim 4 wherein said yeast transcription control sequence is substantially identical to a fragment contained within a 0.6 kb BamHI-Kpnl fragment of the 8kb EcoRI PH05 genomic DNA fragment from chromosome 2 of Saccharomyces cerevisiae.
6. The cloning vehicle of claim 1 wherein said yeast signal sequence codes for a peptide that is substantially identical to the signal peptide expressed by a naturally occurring phosphate-repressible yeast acid phosphatase gene.
7. The cloning vehicle of claim 6 wherein said yeast signal DNA sequence codes for a signal peptide identical to a complete signal peptide expressed by a PHQ5 genomic fragment.
8. The cloning vehicle of claim 1 wherein the three amino acids at the carboxy-terminus of the signal peptide are Ala-Asn-Ala.
9. The cloning vehicle of claim 8 wherein said yeast signal sequence codes for the following signal peptide: Met-Phe-Lys-Ser-Val-Val-Tyr-Ser-Ile-LeuAla-Ala-Ser-Leu-Ala-Asn-Ala.
10. The cloning vehicle of claim 9 wherein the yeast signal sequence (including the ATG translation start codon) is either
ATGTTTAAATCTGTTGTTTATTCAATTTTAGCCGCTTCTTTGGCCAATGCA; or ATGTTTAAATCTGTTGTTTATTCAATTTTAGCCGCTTCTTTGGCCAACGCG
11. The cloning vehicle of claim 1 wherein said exogenous DNA sequence codes for a nonsecretory protein or peptide.
12. The cloning vehicle of claim 1 wherein said exogenous DNA sequence codes for a secretory protein or peptide.
13. The cloning vehicle of claim 1 wherein said exogenous DNA sequence codes for the mature form of alpha hCG.
14. The cloning vehicle of claim 14 wherein said cloning vehicle is 99N-alpha (NRRL No. B 15791).
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK103786A DK103786A (en) | 1984-07-09 | 1986-03-07 | GAER CLONING AID |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62920284A | 1984-07-09 | 1984-07-09 | |
US629,202 | 1984-07-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1986000638A1 true WO1986000638A1 (en) | 1986-01-30 |
Family
ID=24522016
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1985/001310 WO1986000638A1 (en) | 1984-07-09 | 1985-07-09 | Yeast cloning vehicle |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0189463A4 (en) |
JP (1) | JPS61502795A (en) |
DK (1) | DK103786A (en) |
WO (1) | WO1986000638A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5037743A (en) * | 1988-08-05 | 1991-08-06 | Zymogenetics, Inc. | BAR1 secretion signal |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0100561A1 (en) * | 1982-08-09 | 1984-02-15 | Ciba-Geigy Ag | Yeast hybrid vectors and their use for the production of polypeptides |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3382547D1 (en) * | 1983-01-12 | 1992-05-27 | Chiron Corp | SECRETORIC EXPRESSION IN EUKARYOTS. |
-
1985
- 1985-07-09 JP JP50313085A patent/JPS61502795A/en active Pending
- 1985-07-09 WO PCT/US1985/001310 patent/WO1986000638A1/en not_active Application Discontinuation
- 1985-07-09 EP EP19850903627 patent/EP0189463A4/en not_active Withdrawn
-
1986
- 1986-03-07 DK DK103786A patent/DK103786A/en not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0100561A1 (en) * | 1982-08-09 | 1984-02-15 | Ciba-Geigy Ag | Yeast hybrid vectors and their use for the production of polypeptides |
Non-Patent Citations (11)
Title |
---|
ARIMA et al, Nucleic Acids Research Vol. 11, 1983, pages 1657-1672 * |
BAJWA et al, Nucleic Acids Research, Vol. 12, 1984, pages 7721-7739 * |
BOOTHBY et al, CHEMICAL ABSTRACTS Vol. 94, 1981, Abstract No. 100574z of Chorionic Gonadotropin (Proc. Conf.), 1980, pages 253-275 * |
FIDDES et al, CHEMICAL ABSTRACTS, Vol. 95, 1981, Abstract No. 110370q of J. Mol. Appl. Genet, Vol. 1, 1981, pages 3-18. * |
FIDDES et al, Nature, Vol, 281, 1979, pages 351-356 * |
HINNEN et al, CHEMICAL ABSTRACTS, Vol. 102, 1985, Abstract No. 40931k of Found. Biotech. Ind. Ferment. Res., Vol. 1, 1983, pages 157-163. * |
KRAMER, CHEMICAL ABSTRACTS, Vol. 102, 1985, Abstract No. 40980a of EP 124, 874, 14 November 1984. * |
MEYHACK et al, The EMBO Journal, Vol. 1, 1982, pages 675-680. * |
See also references of EP0189463A4 * |
SUNTORY, Ltd., CHEMICAL ABSTRACTS, Vol. 101, 1984, Abstract No. 164746c of JP 59 74, 988, 27 April 1984 * |
THILL et al, Molecular and Cellular Biology, Vol. 3, 1983, page 570-579. * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5037743A (en) * | 1988-08-05 | 1991-08-06 | Zymogenetics, Inc. | BAR1 secretion signal |
Also Published As
Publication number | Publication date |
---|---|
JPS61502795A (en) | 1986-12-04 |
DK103786D0 (en) | 1986-03-07 |
DK103786A (en) | 1986-05-07 |
EP0189463A1 (en) | 1986-08-06 |
EP0189463A4 (en) | 1988-04-18 |
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