Classifications

• • 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-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 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.
• 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' end 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. Summary of the Invention
• the invention features generally a cloning vehicle that is used to transform a microorganism for expression and secretion of a protein.
• the cloning vehicle includes, in order of transcription: a yeast transcription control DNA sequence, a DNA sequence coding for a yeast signal peptide and an exogenous DNA sequence coding for a desired protein.
• the invention features a DNA fragment which includes: a signal sequence substantially identical to a yeast PH05 transcription control sequence; a sequence coding for a signal peptide substantially identical to a complete PH05 signal peptide, and having the same three carboxy-terminal amino acids as a PH05 signal peptide; and a restriction endonuclease site adjacent to the carboxy terminal end of the signal sequence.
• the invention features a method of making the above-described plasmid by providing naturally occurring PH05 transcription control and signal sequences, and modifying them by creating a restriction endonuclease cleavage site at the 3' end of the signal sequence without changing the identity of the three carboxy-terminal amino acids encoded by the DNA signal sequence.
• the cloning vehicle containing the exogenous DNA sequence can be used to transform a yeast host, the transformed host is cultured, and secretion of the desired protein is obtained.
• yeast transcription control DNA sequence means a DNA sequence naturally occurring in yeast which is effective to initiate transcription of DNA.
• yeast signal DNA sequence means a DNA sequence which codes for a signal peptide sequence that naturally occurs in yeast.
• Exogenous DNA refers to DNA which is not naturally found associated with the yeast transcription control DNA sequence and the yeast signal DNA sequence of the vehicle of the invention.
• the transcription control DNA sequence is identical to the transcription control DNA sequence of a naturally occurring yeast gene, and the yeast signal DNA sequence codes for a peptide identical to a signal peptide produced by that gene.
• the yeast gene is the PH05 gene, which codes for the major repressible acid phosphatase enzyme.
• PH05 is contained within an 8-kilobase (kb) EcoRI region of the genomic DNA of chromosome 2 in Saccharomyces cerevisiae. Both the transcription control sequence and the adjacent signal DNA sequence of this gene are contained within a smaller 600-base pair (bp) BamHI-Kpnl portion of this region.
• the exogenous DNA can be yeast DNA coding for nonsecreted (or secreted) yeast proteins; however, preferably, the exogenous DNA sequence corresponds to a sequence coding for a nonyeast protein.
• the above described cloning vehicle enables expression of exogenous DNA in yeast, so that the protein product is secreted by the yeast into the surrounding medium.
• 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 exogenous protein 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 protein 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.
• the above described cloning vector is useful for expression and secretion of DNA sequences coding for any secretory and nonsecretory exogenous proteins. 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 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 PH05 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 PH05 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.
• 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
• the functional TRPl 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 TRPl 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 TRPl 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 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 99N is not important for this function;
• PH05 promoter plus all of the DNA sequence necessary to encode the 17 amino acid signal peptide (including the initial methionine);
• 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.
• 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).
• 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 HincII 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 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 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.
• 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 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
• Bacterial transformants are checked for the presence of a Mlul site; such a plasmid, pAPPM. is isolated.
• pAPPM is digested with Mlul and synthetic
• 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.
• alpha hCG human chorionic gonadotropin
• 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.
• 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 Bal31 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 fragment can be digested with Pstl and synthetic 10-mers (5'CATCAGGAGC3') and 18-mers
• 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 Sad with the recognition sequence GAGCTC. This has been achieved by Iigating 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 Sad 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.
• Plasmid 99N-alpha has been deposited with the
• 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:
• 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 S -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/1 KCl.
• This medium is called LP (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 antigenically active alpha hCG was found in the culture medium. Typical levels were 0.2 mg/1 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. What is claimed is:

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• 1985
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