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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
• • 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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
  • C07K14/56—IFN-alpha
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  • 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
  • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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  • 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
• • 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
  • C07K2319/70—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

• the 5'-promoter region, translation start and signal peptide sequence of the human leukocyte interferon gene were replaced with those from the yeast alcohol dehydrogenase (ADH1) gene.
• ADH1 yeast alcohol dehydrogenase
• the ability to control the activity of the promoter is highly useful to permit the attainment of high levels of interferon expression. It has now been discovered tha t the promoter for the S UC2 invertase gene is controllable by a mechanism of passive regulation in the production of interferon in a yeast cel l.
• a still further object of the present invention is to obtain living cells which are capable of producing a polypeptide product by a method wherein the synthesis of the polypeptide is passively regulated by a level of material in culture medium for the cells.
• a still fu rther object of the present inven tion i s to provide genetic recombinant mater ial carry ing a pr omoter of the yeast SUC2 invertase gene .
• yeast cells capable of producing polypeptide products such as interferon obtained therefrom at a preselected stage of cell growth whereby the cells carry a passive promoter.
• the cells each contain the promoter in operative relationship to a gene, the relationship of which is not found in nature.
• the promoter is introduced into the cells or ancestors of the cells for the purpose of permitting a passive signal to turn on synthesis of a desired polypeptide product by expression of the gene upon activation of the promoter after a period of promoter inactivity during cell growth.
• the expression of the gene for human leukocyte interferon is controlled by the SUC2 promoter of the yeast strain Saccharomyces cerevisiae.
• the SUC2 promoter is a DNA segment that contains the transcription start signal for the structural gene for invertase.
• the modified yeast strain of the present invention displays an unprecedented mode of regulation of the expression of the interferon gene in yeast. Synthesis of the interferon is repressed when the yeast are grown in glucose medium, and derepressed when glucose is depleted. No yeast strain previously described shows regulation of interferon synthesis over a broad range by changes in the culture medium.
• a DNA segment which contains a SUC2 promoter linked to an interferon gene for directing the expression of the gene within a yeast cell.
• the segment is preferably a
• a SUC2 promoter of invertase in yeast is inserted before the interferon gene in a chromosome or plasmid in such a fashion that the plasmid or chromosome is replicated and carried by the cell as part of its genetic information.
• interferon IFN
• the regulation of the SUC2 promoter by glucose permits propagation of the yeast without the potentially deleterious effects of IFN production, since overly high levels of IFN may be toxic to cells.
• the glucose regulation of the SUC2 promoter affords maximal synthesis of IFN at low glucose concentration.
• -construction of a yeast strain with these properties is particularly desirable for commercial production of IFN because of existing large-scale yeast fermentation technology and also because of the low toxicity of S. cerevisiae.
• Microorganisms prepared by the genetic engineering processes described herein are exemplified by cultures now on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland. These cultures are identified by Accession Number 20644, Strain Designation CGY144 and were deposited by Collaborative Research, Inc.
• a particular DNA segment is linked to an interferon gene and incorporated in a modified strain of Saccharomyces cerevisiae so that it produces human leukocyte interferon (LelFN) under the control of the SUC2 promoter of the yeast invertase gene.
• the S. cerevisiae is genetically transformed with a novel recombinant DNA plasmid.
• the plasmid was constructed by ligation of DNA segments from the E. coli plasmid pBR322, yeast genomic and plasmid DNA's and synthetic DNA linkers.
• the construction of plasmid pBR322, sequenced by J. G. Sutcliffe, Cold Spring Harbor Symposium 43, 77-90 (1979) is shown diagrammatically in Table 1.
• plasmid for joining with the exogenous gene
• a wide variety of techniques can be used, including the formation of or introduction of cohesive termini. DNA molecules with blunt ends can also be joined together.
• Cohesive termini may be produced in a variety of ways; the plasmid and gene may be cleaved in such a manner that the two chains are cleaved at different sites to leave extensions at each end which serve as cohesive termini.
• Cohesive termini may also be introduced by removing nucleic acids from the opposite ends of the two chains or alternatively, introducing nucleic acids at opposite ends of the two chains. Methods which may be employed in joining cleaved DNA segments depend on the nature of the termini, as described below.
• Blunt-ended DNA's are produced as for example, by cleavage with any of a number of restriction endonucleases, such as Haelll.
• restriction endonucleases such as Haelll.
• random shear breakage or a restriction enzyme making staggered cuts such as EcoRI, Hindlll, or BamHI, may be used, but the DNA termini must then be made blunt by biochemical methods.
• biochemical methods include incubation with single-strand-specific nuclease S1, as described in the following articles:
• blunt termini can be created by incubation with T4 DNA polymerase [see Stakura, K., Hirose,
• linkers which are short duplexes containing a restricton endonuclease cleavage site. Such linkers are joined to DNA by T4 DNA-ligase catalyzed blunt-end joining. After digesting the product with the restriction enzyme that cleaves within the linker, the DNA is terminated with cohesive ends.
• T4 DNA-ligase catalyzed blunt-end joining After digesting the product with the restriction enzyme that cleaves within the linker, the DNA is terminated with cohesive ends.
• Linker refers to a duplex, blunt-ended DNA molecule from 6-14 base pairs in length, containing the recognition site for a restriction endonuclease that produces cohesive or blunt termini.
• the plasmid serves as the vehicle for introduction of the foreign gene into the yeast cell.
• a plasmid it is not necessary to use a plasmid, since any molecule capable of replication in yeast can be employed.
• the DNA molecule can be attached to a vector other than a plasmid, which can be a virus or cosmid as is known in the art; or it can be integrated into the chromosome.
• the recombinant plasmid or plasmid chimera is constructed in vitro. Since the annealing and ligation process not only results in the formation of the recombinant plasmid, but also in the recircularization of the plasmid vehicle, a mixture of ligation products i s obtained involvi ng the orig inal plasmid and the foreign DNA. Only the or iginal plasmid and the DNA chimera cons isting of the plasmid vehicle and linked foreign
• DNA will normally be capable o f replication.
• replication of both the plasmid vehicle genotype and the foreign genotype will occur.
• the transformation of the bacterial cells will result in a mixture of bacterial cells, the dominant proportion of which will not be transformed. Of the fraction of cells which are transformed, some significant proportion, but in some cases a minor proportion, will have been transformed by the recombinant plasmid . In any event , only a very small fraction o f the total number of cells which are present will have the desired phenotypic characteristics.
• a selectable genetic marker is included on the original plasmid, such as resistance to an antibiotic or heavy metal.
• the cells can then be grown on an agar medium containing the growth inhibiting substance. Since E. coli is used as the bacteria for transformation in the present invention, ampicillin is used as the growth inhibiting material to afford selection in E. coli. Only available cells having the resistant genotype will survive. If the foreign gene does not provide a phenotypical property, which allows for distinction between the cells transformed by the plasmid vehicle and the cells transformed by the plasmid chimera, a further step is necessary to isolate the replicated plasmid chimera from the replicated plasmid vehicle.
• the steps include lysing of the cells and isolation and separation of the DNA by conventional means or random selection of transformed bacteria and characterization of DNA from such transformants to determine which cells contain molecular chimeras. This is accomplished by physically characterizing the DNA by electrophoresis, gradient centrifugation, sequence analysis or electron microscopy.
• DNA may then be analyzed in a variety of ways.
• One way is to treat the plasmid with an appropriate restriction enzyme and analyze the resulting fragments for the presence of the foreign gene. Other techniques have been indicated above.
• the E. coli may be grown and multiplied and the recombinant plasmid employed for transformation of the S. cerevisiae strain.
• SUC2 promoter as employed in the present invention, also designated P SUC2 , is a 0.9 kilobase DNA sequence from the yeast genome that contains the promoter for the major, regulated messenger RNA (mRNA) transcript of the invertase gene whose protein is secreted and glysoylated as opposed to the intracellular, nonglycosylated invertase.
• mRNA messenger RNA
• yeast RNA transcription initiates within 100 base pairs of the right end of the sequence and proceeds rightward. See M.
• the interferon gene referred to which can be promoted by the promoter used in this invention, is any one of the three classes of interferon genes described below:
• leukocyte - derived from leukocyte or lymphoblastoid cells designated LelFN or IFN- ⁇ ;
• fibroblast - derived from fibroblast cells designated FIFN or IFN- ⁇ ;
• IFN- ⁇ immune - derived from mitogen- or antigen-stimulated lymphoid cells, designated IFN- ⁇ .
• human leukocyte interferon is enhanced compared to levels of production in prior methods, and with the use of the S. cerevisiae strain a new human leukocyte interferon is produced.
• the yeast strain employed as the host cell in the preferred embodiment of the present invention is
• Saccharomyces cerevisiae a common laboratory strain of yeast used for its low toxicity and well-known genetic characteristics. This strain is readily cultivatable on a large scale.
• the recombinant DNA material of the present invention containing the SUC2 promoter can be used to express interferon in any yeast cells capable of transformation.
• Saccharomyces cerevisiae is a yeast whose vegetative reproduction occurs by multilateral budding cells. Such cells are usually found in pairs or small clusters. The species is usually diploid where spores are produced directly in vegetative cells, but the species can also grow as haploid. In addition, S. cerevisiae forms an ascus with one to four spheroidal spores in each ascus. The ascus for this species does not rupture at maturity. The yeast has a strongly fermentative as well as respiratory metabolism. Selected strains are referred to as distillers' yeasts and baker's yeast.
• yeasts can be cultivated under relatively uniform conditions on common laboratory media.
• the usual growth requirements of yeast include:
• yeast nitrogen base obtained from Difco
• YNB yeast nitrogen base
• the nitrogen source is ammonium sulfate.
• the desired carbon source must be added and is normally at a concentration of 0.5 - 3% . Additions are made to this medium to fit particular strain requirements.
• the pH range of the medium is usually from pH 3 - 8. The preferred range is pH 4.5 - 6.5.
• This construction consists of several components generally used in "shuttle" vectors, i.e., plasmids that can be maintained either in E. coli or yeast.
• the plasmid described in Table 4 is a modified construction of plasmid YIp5, as described by K. Struhl, D. T. Stinchcomb, S. Scherer and R. W. Davis, Proc. Nat. Acad. Sci. USA 76, 1035-1039 (1979) [see Table 2].
• Segment (1) is a 2.4 kilobase fragment of plasmid pBR322, and contains a DNA replication origin and the ⁇ -lactamase gene, allowing propagation of the DNA in E. coli and continuous selection for its presence by ampicillin resistance.
• Segment (2) is a 1.6 kb Hpal to Hindlll fragment of the yeast 2 ⁇ plasmid containing an initiation site for replication in yeast.
• the 2 ⁇ plasmid is described by J. L. Hartley and J. E. Donelson, Nature 286, 860-865 (1980) .
• Segment (3) is a 1.1 kb DNA containing the yeast URA3 gene which encodes the enzyme orotidine-5' monophosphate decarboxylase that catalyzes an essential step in uracil biosynthesis in yeast.
• the URA3 gene is described by M. Bach, F. Lacroute, and D. Botstein, Proc. Nat. Acad. Sci.
• Segment (3) is inserted at the Aval site of plasmid pBR322, thus dividing this region of pBR322 into Segments (6) and (7) .
• the unique aspect of this construction is the inclusion of DNA segments (4) and (5).
• Segment (4) is a 0.9 kb EcoRI to Hindlll DNA fragment of the yeast genome that contains the regulated promoter for the major messenger RNA transcript of the invertase gene. In yeast, RNA transcription initiates within 100 base pairs of the right end of segment (4) and proceeds rightward (as shown in Table 4).
• Segment (5) contains the gene for the new human leukocyte IFN in its mature form, oriented such that transcripts originating from the SUC2 promoter copy the gene into mRNA coding for interferon.
• the interferon coding sequence is not the full sequence of human LelFN mRNA, but one in which the sequence coding for the signal peptide of the interferon precursor has been replaced by a translation initiation codon directly preceding the sequence coding for the mature secreted protein for the new human leukocyte interferon.
• Codon (a) is the initiating methionine codon and (b) is the initial cysteine codon of the new human LelFN.
• the construction also contains the cleavage site of restriction endon ⁇ clease Clal.
• the synthetically produced sequence shown above may exist in nature in random fashion.
• Segment (5) is joined to Segment (6) of pBR322 DNA by a HindIII linker.
• This plasmid vector is maintained in multiple copies per cell in a strain of Saccharomyces cerevisiae (CGY 123) that has the genotype MATa leu2-3 leu2-112 ura3-50. Since this host is a uracil auxotroph, only those cells containing the plasmid with the URA3+ gene can grow in minimal medium without uracil. This selection reduces the accumulation of yeast cells that have lost the plasmid by segregation during cell division.
• the synthetically produced sequence shown above may exis t in nature in r andom f ash ion.
• the essential feature in the plasmid that must be maintained is the orientation of the SUC2 promoter relative to the IFN gene to be transcribed. Permissible modifications in the plasmid DNA would include:
• the yeast strain described herein produces a new human
• LelFN when cultured in a medium with low glucose levels.
• the yeast are propagated in a medium containing 6.7 g/l yeast nitrogen base, 50 mg/l L-leucine and 2% glucose.
• the yeast cells are removed from the high glucose medium by centrifugation, and resuspended in minimal medium containing 0.05% glucose. In 2 to 3 hours, the SUC2 promoter is fully active and the yeast accumulate
• the cells are harvested by centrifugation.
• the cell-free extract containing LelFN is obtained by breaking open the yeast cells by vigorous vortexing with glass beads in a solution of 0.1 M Tris-HCl, pH8, 20% glycerol, and ImM
• Sendai virus induced lymphocytes were disrupted by means of a Dounce homogenizer into 40 ml of cold buffer (10° C) consisting of 50 mM NaOAc, pH 5.2; 6 M guanidine HCl; and 0.1 M 2-mercaptoethanol.
• the resulting solution was sonicated at 60W pulsed power for 2x30 seconds and then layered onto 3 ml shelves of 5.8 M CsCl, pH 7.2, containing 0.1 M EDTA.
• the material was centrifuged at 15° C in a Beckman Type 50 Ti rotor at 40,000 rpm overnight.
• the pellet was resuspended on ice for 20 minutes in 6.6 ml of the above cold buffer plus 20 mM EDTA, and then treated with 3.3 ml of ice-cold absolute ethanol. After 1 hour at -20° C, the precipitate was pelleted by a centrifugation at 8,000 rpm for 20 minutes at -10° C. The pellet was resuspended two times in 18 ml of the preceding buffer, treated with 9 ml of ice cold absolute ethanol, chilled one hour at -20° C and the pellet collected as decribed previously.
• the final pellet was resuspended in 8 ml of 0.1 M EDTA with heating at 60° C, and then 0.1 volume of 2M NaOAC, pH 5.0, and 2 volumes of ice-cold absolute ethanol were added and the solution placed at -20° overnight.
• the RNA precipitate was collected by centrifugation at 8,000 rpm for 20 minutes at -10° C, and was dissolved in 5 ml water. The yield was 396 mg RNA.
• the RNA solution was diluted with 5 ml of 2x concentrated binding buffer (20 mM Tris-HCL, pH 7.5; 2mM EDTA, pH 7.0; 0.4% SDS; and 0.24 M NaCl).
• RNA was applied to a 1 ml oligo-dT-cellulose column, the column was washed with Ix concentrated binding buffer and then the poly A-containing RNA (mRNA) was eluted by washing the column with binding buffer containing no NaCl. About 39 mg of poly A-containing RNA was obtained.
• a portion of the poly A-containing RNA was translated in vitro in a rabbit reticulocyte lysate system [Pelham, H.R.B. and Jackson, R.J. , Eur. J. Biochem. 67, 247-256 (1976)] to confirm the isolation of mRNA coding for interferon.
• cDNA was synthesized from 25 ⁇ g of the lymphocyte poly A-containing RNA by incubation for one hour at 42° C in 50 mM Tris-Hcl, pH 8.3; 100 mM KCl; 8mM MgCl 2 ; 0.4 mM dithiothreitol; 1.2 mM each dATP, dGTP and dTTP; and
• the reaction was terminated by addition of EDTA to 10 mM, and Tris-HCl, pH 8.3, to 200 mM, and the mixture applied to a Biogel A-150m column (0.7 cm x 35 cm) equilibrated and eluted with 10 mM Tris -HCl, pH 7.5, 250 mM NaCl and 1 mM EDTA.
• the peak fractions (0.5 ml each) of large molecular weight DNA were pooled and ethanol precipitated by addition of 1/10 volume 2 M NaOAC, pH 5, and 2.5 volumes cold absolute ethanol.
• the S1-treated double-stranded cDNA (0.21 ⁇ q) was incubated in buffer (60 mM Tris-HCl, pH 7.5; 8 mM MgCl; 5 mM dithiothreitol, 1 mM ATP and 1 mM of each deoxynucleoside triphosphate) with 9 units of E. coli DNA polymerase I at 10° C for 10 minutes and then placed on ice.
• This blunt-ended double stranded cDNA was next incubated in 65 mM Tris-HCl, pH 7.5; MgCl 2 ; 5 mM dithiothreitol; 1 mM ATP, with 160 pmoles of 32 P-labelled Hindlll synthetic linker
• the ligation reaction contained 60 mM Tris-HCl, pH 7.5; 6 mM MgCl 2 ; 7 mM dithiothreitol; 1.2 ⁇ g double-stranded cDNA; 1.2 ⁇ g CGF4 DNA;
• the plaques were transferred to nitrocelluloses and probed as described by Benton and Davis [Benton, W. D. and Davis, R. W. , Science 196, 180-182 (1977] using a 32 P-labelled synthetic oligonucleotide (with the sequence, CATGATTTCTGCTCTGAC, Collaborative Research, Inc.) which corresponds to a known segment of LelFN.
• the oligonucleotide (1 ⁇ g) was kinased with 0.5 mC ⁇ - 32 P-ATP using 6 units of
• T4 polynucleotide kinase (P-L Biochemicals) in a 20 ⁇ l reaction containing 66 mM Tris-HCl, pH 7.5, and 10 mM MgCl 2 .
• the phage which hybridized intensely to the synthetic oligonucleotide probe were picked from the plates and stored in TY medium at 4° C.
• phage DNA' s which has an insert of about 1200 base pairs (bP) was chosen for further study.
• the DNA insert was sequenced by the method of Maxam and Gilbert [Maxam, A. M. and Gilbert , W. , Methods in Enzymol 68 , 499-560 (1980) ] .
• a plasmid , pCGS84 designed to facilitate obtaining expression of LelFN in yeast was cons felted.
• an ATG initiation codon was incorporated at the 5 ' -s ide of the f irst codon (TGT for cysteine) of mature , processed IFN.
• TGT for cysteine
• an oligonucleotide ACACATCGATGTGT which is recognized by Clal and also contains the ATG-TGT sequence was synthesized by Collaborative Research, Inc .
• a Sau3Al fragment which codes the amino acid residues 2 to 61 was purified by digesting 30 ⁇ g of the Hindlll 1.2 kilobase fragment with 10 units Sau3Al restr iction endonuclease in a 50 ⁇ l reaction volume containing 10 mM Tris-HCl, pH 7.5 ; 10 mM MgCl 2 ; and 60 mM NaCl for 4 hours at 37 ° C.
• the DNA fragment was purified by polyacrylamide gel electrophoresis. The DNA was phenol extracted and precipitated with ice-cold absolute ethanol.
• restriction cut plasmid was phenol extracted, ethanol precipitated and treated with calf intestinal phosphatase by the method of H. Goodman and R. J. MacDonald
• Transformation-competent E. coli strain CGE43 (LG90; F- (lac-pro)xlll) was prepared as described previously for CGE6, and 5 ⁇ l of the ligated DNA was mixed with 200 ⁇ l of the cells for 30 minutes at 0° C, heat treated at 37° C for 2 minutes, incubated at 18° C for 10 minutes, and diluted five-fold with fresh tryptone broth. After incubation for 30 minutes at 37° C with shaking, cells were plated on tryptone plates containing ampicillin (20 ⁇ g/ml). Ampicillin-resistant colonies were picked, and the plasmid DNA was prepared and analyzed by restriction enzyme digestion. By these criteria several cells carried the desired plasmid, pCGE32.
• Plasmid pCGE32 DNA (10 ⁇ g ) was cut with the restriction endonuclease HindIII (Collaborative Research, Inc., 12 units) for 2 hours at 37° C in a 20 ⁇ l reaction containing 10 mM Tris-HCl, pH 7.5; 10 mM MgCl 2 ; 60 mM NaCl; and 1 mg/ml bovine serum albumin).
• This DNA was next digested with the endonuclease EcoRl (Collaborative Research, Inc., 15 units) for 3 hours at 37° C in a 20 ⁇ l reaction containing 100 mM Tris-HCl, pH 7.6; 10mM MgCl 2 ; 30 mM NaCl; and 1 mg/ml bovine serum albumin.
• the restriction cut DNA was phenol extracted, ethanol precipitated, redissolved in water and applied to a preparative horizontal 1.5% agarose gel. After electrophoresis for 2 to 3 hours in 40 mM Tris-acetate, pH 7.2, the gel was stained with ethidium bromide and examined under long wavelength ultraviolet light.
• the digested Hindlll to EcoRl band which codes the ATG-TGT to amino acid residue 37 was excised and the DNA extracted by freezing and thawing the gel pieces [Thuring, et al. , Anal. Biochem 66, 213 (1975)].
• the DNA fragment was ethanol-precipitated and redissolved in water.
• the plasmid (20 ⁇ g) containing the IFN clone was cut with the restriction endonuclease Hindlll (New England Biolabs, 180 units) for 2 hours at 37° C as above and then the DNA
• DNA was mixed wi th 100 ⁇ l of the cells for 30 minutes a t 0 °
• the 1.1 kilobase HindIII fragment containing the gene for LelFN was isolated from plasmid pCGE38 (30 JULq) by cutting the plasmid with restricton endonuclease HindIII for 1.5 hours at 37° C as above.
• the restriction cut DNA was phenol extracted, ethanol precipitated, redissolved in water and applied to a preparative 1% agarose gel. After electrophoresis in 40 mM Tris-acetate, pH 7.2, the gel was stained with ethidium bromide and examined under long wavelength ultraviolet light.
• the 1.1 kilobase band was excised and the DNA extraced by freezing and thawing the gel pieces [Thuring, et al. , Anal. Biochem.
• the plasmid, pCGS40 comprises most of YIp5 containing a DNA replication origin and ⁇ -lactamase gene for selection in E. coli, with a 1.6 kilobase fragment of the yeast 3 ⁇ plasmid containing an initiation site for replication in yeast, with a 1.1 kilobase fragment from the yeast chromosomal DNA carrying a URA3 gene for selection in yeast and with a 0.9 kilobase fragment from yeast chromosomal DNA containing the SUC2 promoter of the yeast invertase gene.
• the plasmid pCGS40 was constructed by first cutting 60 ⁇ q of plasmid pRB118 [Carlson, M. and Botstein, D., Cell 28, 145-154
• the 0.9 kilobase DNA fragment containing the SUC2 promoter was placed on the plasmid YIp5 (a shuttle vector which can be selected for and maintained in yeast due to the presence of the URA3 gene or E. coli due to the presence of the Amp gene) [Struhl, D. , Stinchcomb, D. T. , Scherer, S. and Davis, R. W., Proc. Nat. Acad. Sci. USA 76, 1035-1039 (1979)].
• the resulting plasmid, pCGS46 obtained after ligation and transformation (as described above) was purified and its structure verified by analysis with restriction endonucleases.
• the plasmid, pCGS40 was the result of cutting the plasmid pCGS46 with restriction endonuclease PvuII for 1 hour at 37° C.
• the resulting plasmid, pCGS40 can be grown and its presence can be selected for in either E. coli or Saccharomyces cerevisiae.
• the yeast strain CGY123 (MATa, leu 2-3, leu 2-112, ura 3-50) was transformed with the plasmid DNA by the method of A. Hinnen, J. B. Hicks, and G. Fink [Hinnen, A., Hicks, J. B. and Fink, G. F. , Proc. Nat. Acad. Sci. USA 75, 1929-1933 (1978)].
• Yeast transformants CGY144 capable of growth without added uracil due to the presence of URA3 gene on the plasmid, were picked and the plasmid DNA pCGS84 was prepared following amplification of these transformants.
• DNA was analyzed by restriction enzyme digestion with restriction endonucleases HindIII and EcoRl and the proper
• yeast cells were grown at 30° C with agitation in a medium containing 6.7 g/l yeast nitrogen base, 30 mg/l
• the cells were collected by centrifugation, resuspended in 0.25 ml 0.05 M Tris-HCl, pH 7.6, 20% glycerol and 1 mM PMSF, and frozen at -20° C.
• the cells were disrupted by glass beads by the method of M Rose, et al. [Rose, M., Casadaban, M. J. and Botstein, D., Proc. Nat. Acad. Sci. USA 78, 2460-2464 (1981)] and the amount of interferon activity in the cellular extract was determined by conventional methods to be 10 units/l.
• the sequencing information for the human leukocyte interferon gene produced is shown in Table 5.
• the present..invention is mainly concerned with the production of human leukocyte interferon of a particular type.
• other interferons can be obtained and expressed using the SUC2 promoter of this invention in the operative relationship defined.
• the ability to use a passive signal to initiate expression of a desired polypeptide such as interferon is important for production of polypeptide products in large volume.
• expression can be obtained without introduction into a growing culture of an external inducer to stimulate expression.
• the stimulus can be depletion of glucose as in the present example, whereupon the culture, as a self-contained system, will initiate expression in high quantities of a desired polypeptide.
• Such polypeptides may be enzymes or other biologically active proteins.
• the promoters may also differ.
• the present example is but one example of passive production.
• the SUC2 promoter has been linked to genes other than interferon to promote their expression in yeast. Thus far, comparative studies show the levels of expression of interferon to be far greater than those of the few other genes tested. It is expected that SUC2 can promote the expression of a variety of genes at levels high enough for commercial applications.

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