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  • 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
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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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
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  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
  • C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
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  • 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
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  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
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  • 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 present invention is directed to novel DNA constructs containing at least the translated signal peptide portion of the Saccharomvces cerevisiae BARl gene and at least one structural gene foreign to a host cell transformed with said construct. Transformation of host organisms by such constructs will result in expression of a primary translation product comprising the structural protein encoded by the foreign gene fused to the signal peptide of BARl so that the protein is processed through the host cell secretory pathway and may be secreted from the host cell into the culture medium or the periplasmic space.
• Various procaryotic and eucaryotic microorganisms have been utilized as hosts for production of heterologous polypeptides, i.e., polypeptides which are not naturally produced by the host, by way of re ⁇ ombinant DNA methodology.
• Various eucaryotic fungal species are of particular interest, including Saccharomvces cerevisiae, Schizosaccharomyces pombe, Aspergillus and Neurospora.
• Saccharomvces cerevisiae Saccharomvces cerevisiae
• Schizosaccharomyces pombe Aspergillus and Neurospora.
• Much work has been done in the budding yeast S. cerevisiae.
• Yeast cells when transformed with a suitable DNA construct, such as a plasmid, have been made to express heterologous genes contained in the plasmid.
• S. cerevisiae is known to secrete some of its naturally produced proteins, although knowledge of the process is quite limited compared to what is known about secretion of proteins from bacteria and mammalian cells. It appears that most of the secreted yeast proteins are enzymes which remain in the periplasmic space, although the enzymes invertase and acid phosphatase may also be incorporated into the cell wall.
• the proteins which are known to be secreted into the culture medium by S. cerevisiae include the mating pheromones (o-factor and a-factor), killer toxin, and the protein accounting for Barrier activity (hereinafter "Barrier"). Secretion through the cell wall into the medium is also referred to as "export". S.
• ⁇ -factor comprising a precursor polypeptide that is cleaved from mature ⁇ -factor
• non-translated gene sequences including promoter and regulatory regions
• Another object of the present invention is to provide a method for producing foreign proteins which are secreted from a microbial host.
• the present invention is directed to DNA constructs and methods for using same, which constructs comprise at least the signal peptide coding sequence of the Saccharomyces cerevisiae BARl gene, at least one structural gene foreign to the host organism, and a promoter which controls the expression in a host organism of a fusion protein comprising the BARl signal peptide and the foreign protein.
• FIG. 3 illustrates the construction of plasmid p254
• the structural BARl gene, or a portion thereof, and the structural gene to be expressed will preferably be under control of a single promoter. Methods of ligation of DNA fragments are amply described and are well within the ability of those with ordinary skill in the art to perform.
• the DNA coding sequence of the protein to be expressed may be essentially that of any protein, particularly proteins of commercial importance, such as interferons, insulin, proinsulin, ⁇ -1-antitrypsin, growth factors, and tissue plasminogen activator.
• the different steps can be conveniently done in microtiter dishes, until the pellets are transferred to the scintillation vials.
• A. recombinant plasmid pool comprising the entire yeast genome was constructed (Nasmyth and Tat ⁇ hell, Cell 19: 753-764, 1980) using the shuttle vector YEp13 (Broach et al., Gene 8: 121-133, 1979). Yeast DNA fragments, produced via a partial Sau 3A digestion, were inserted into Bam Hi-digested YEp13.
• the plasmid pool was used to transform S . cerevisiae strain XP635-10C (MATa leu2-3 leu2-112 bar1-1 ga12: ATCC #20679) and transformants were selected for leucine prototrophy and growth on a concentration of ⁇ -factor that is inhibitory to the a barl cells. Resultant colonies were then screened for the ability to secrete Barrier activity. Two colonies were found which carried both leucine independence and the ability to secrete Barrier. These colonies carried the plasmids designated pBAR2 and pBAR3.
• pBAR3 plasmid insert comprised a portion of the insert of pBAR2, but oriented in the vector in the opposite direction.
• Further subcloning and screening for Barrier secretion localized the functional BARl gene sequence to a region of approximately 2.75 kb. This fragment comprises the coding sequence, nontranslated transcribed sequences, promoter, regulatory regions, transcription terminator, and flanking chromosomal sequences.
• the plasmid pBAR2 was digested with restriction endonucleases Hind III and Xho I and a fragment of approximately 3 kb was purified by electrophoresis through an agarose gel. This fragment was inserted into plasmid pUC13 which had been digested with Hind III and Sal I.
• the resulting recombinant plasmid, desig nated pZV9 (FIG. 2), can be used to transform E. coli, but lacks the necessary origin of replication and selectable marker for a yeast vector.
• the plasmid pZV9, in a transformant strain of E. coli RRI has been deposited with ATCC under accession no. 53283.
• fragments of the BARl gene comprising the 5' regulatory region and a portion of the coding sequence were used.
• the fusion of the BARl and proinsulin gene fragments was made in the proper reading frame and at a point in the BARl sequence at which the resulting fusion polypeptide may be cleaved, preferably in vivo.
• Several sites in the BARl gene are potential cleavage sites; the Arg-Arg at position 177-178 was selected as the test site of fusion with proinsulin.
• the 5' regulatory sequences and approximately 800 bp of the coding sequence of BARl were purified from plasmid pZV9 as a 1.9 kb Hind Ill-Sal I fragment.
• FIG. 3 there is shown a method for subcloning human preproinsulin cDNA.
• a human preproinsulin cDNA (preBCA clone), p27, is produced by G-tailing Pst I digested pBR327 and inserting C-tailed DNA, made by reverse transcribing total RNA from human pancreas. Plasmid pBR327 is described by Soberon et al., Gene 9: 287-305 (1980) and the sequence of human preproinsulin is reported by Bell et al., Nature 232: 525-527 (1979). The complete translated sequence was cut out as a Nco I-Hga I fragment.
• plasmids were screened for the presence of Eco RI, Nco I and Xba I sites flanking a 340 bp insert.
• a plasmid having these properties is termed p47, shown in FIG. 3.
• a proinsulin (BCA) fragment with a blunt 5' end was generated by primer repair synthesis (Lawn et al., Nuc. Acids Res. 9: 6103-6114, 1981) of plasmid p47.
• Subsequent digestions with Xba I yielded a 270 bp fragment which was inserted into pUC12.
• the vector was prepared by cutting with Hind III, blunting the ends with DNA polymerase I (Klenow fragment), cutting with Xba I, and gel purifying.
• the resultant vector fragment comprising a blunt end and a Xba I sticky end, was ligated to the above described BCA fragment.
• GGTCGACC T4 polynucleotide kinase and ⁇ - 32 P-ATP and were ligated to the blunted proinsulin fragment.
• the proinsulin fragment and the 1.9 kb BARl fragment were ligated together into pUC13 which had been digested with Hind III and Bam HI. This construct was used to transform E. coli K12 (JM83).
• Transformed cells were screened for ampicillin resistance and production of white colonies. Further screening by restriction endonuclease digestion using Hind III, Bam HI, and Sal I identified a plasmid (pZV27) containing a Hind III-Bam HI fragment of the proper size and a single Sal I site.
• plasmid pZV27 was digested with Hind HI and Bam HI and the ca. 2.2 kb BARl-proinsulin fusion fragment was gel purified. This fragment was then inserted into the replicative form of the phage vector M13mpll (Messing, Meth.
• kinased primer 7.5 pmol was then combined with 80 ng of M13 sequencing primer (Bethesda Research Laboratories, Inc.) This mixture was annealed to 2 ⁇ g of single stranded mpll-ZV29 and the second strand was extended using T4 DNA ligase and DNA polymerase I (Klenow fragment), as described for oligonucleotidedirected mutagenesis (two primer method) by Zoller et al. (Manual for Advanced Techniques in Molecular Cloning Course, Cold Spring Harbor Laboratory, 1983). DNA prepared in this way was used to transfect E. coli K12 (JM103) and plaques were screened using the kinased oligomer as probe (Zoller et al..
• Plaques so identified were used for preparation of phage replicative form (RF) DNA (Messing, ibid.). Restriction enzyme digestion of RF DNA identified two clones having the proper Xba I restriction pattern (fragments of 7.5 kb, 0.81 kb, and 0.65 kb) and lacking a Sal I restriction site (which was present in the deleted region of the BARl-proinsulin fusion).
• RF phage replicative form
• the S . cerevisiae alcohol dehydrogenase I promoter (hereinafter ADHI promoter; also known as ADCI promoter) was tested for use in directing expression of foreign polypeptides in conjunction with BARl sequences.
• a plasmid comprising these sequences was constructed.
• the plasmid pZV50 (FIG. 5B) comprises the S . cerevisiae ADHI promoter, the BARl-proinsulin fusion described above, and the terminator region of the S . cerevisiae triose phosphate isomerase (TPI1) gene (Alber and Kawasaki, J. Molec. Appl. Genet. 1 : 419-434, 1982). It was constructed in the following manner. Referring to FIG.
• plasmid pAH5 (Ammerer, ibid.) was digested with Hind III and Bam HI and the 1.5 kb ADHI promoter fragment was gel purified. This fragment, together with the Hind III-Eco RI polylinker fragment from pUC13, was inserted into Eco RI, Bam Hl-digested pBR327, using T4 DNA ligase. The resultant plasmid, designated pAM5, was digested with Sph I and Xba I, and the approximately 0.4 kb ADHI promoter fragment was purified on a 2% agarose gel.
• the TPIl terminator was obtained from plasmid pFGl (Alber and Kawasaki, ibid.). pFGl was digested with Eco RI, the linearized plasmid ends were blunted using DNA polymerase I (Klenow fragment), and Bam HI linker sequences (CGGATCCA) were added. The fragment was digested with Bam HI and religated to produce plasmid pl36. The 700 bp TPIl terminator was purified from p136 as a Xba I-Bam HI fragment.
• This fragment was inserted into Xba I, Bam Hl-digested YEp13, which was then cut with Hind III, the ends blunted using DNA polymerase I (Klenow fragment), and religated to produce plasmid p270.
• the TPIl terminator was purified from p270 as a Xba I-Bam HI fragment, and was inserted into Xba I, Bam Hl-digested pUC13 to yield plasmid ml15.
• Plasmid pZV45 was subsequently digested with Sph I and Bam HI, and the ADHI-BARl- proinsulin-TPI terminator sequence was gel purified. This fragment was inserted into YEp13 which had been digested with Sph I and Bam HI, to produce the S . cerevisiae expression vector pZV50.
• the S. pombe ADH promoter was obtained from a library of DNA fragments derived from S. pombe strain 972h- (ATCC 24843), which had been cloned into YEp13 as described by Russell and Hall (J. Biol. Chem. 258: 143-149, 1983).
• the promoter sequence was purified from the library as a 0.75 kb Sph I-Eco RI fragment. This fragment and the Eco RI-Hind III polylinker fragment of pUC12 were ligated into YEp13 which had been digested with Sph I and Hind III. The resulting plasmid is known as pEVP-11.
• the ADH promoter was purified from pEVP-11 as a Sph I-Xba I fragment.
• Plasmid pZV33 was digested with Xba I and Bgl II and the ca. 340 bp BARl fragment, which includes the ATG initiation codon, was purified.
• pZV33 was digested with Bgl II and Sst I, and the BARl-proinsulin fusion sequence was purified. The three fragments were combined with Sph I, Sst I-digested pUC18 to produce plasmid pZV46. As pUC18 is not effective for transformation of S.
• the BARl signal peptide was tested for its ability to direct the export of ⁇ -factor from a yeast transformant.
• Several plasmids containing DNA fragments coding for fusion proteins with varying lengths of the BARl protein and 1 or 4 copies of mature ⁇ -factor were constructed. These plasmids were transformed into an a/ ⁇ diploid host strain and the transformants assayed for ⁇ -factor production by the halo assay.
• Plasmids pSW94, pSW95, pSW96, and pSW97 comprise the S. cerevisiae triose phosphate isomerase (TPIl) promoter, a 355 bp or 767 bp fragment of the BARl gene (comprising 114 or 251 codons of the 5' end of the BARl coding sequence, respectively) and either one or four copies of the alpha-factor (MF ⁇ 1) coding sequence.
• TTIl triose phosphate isomerase
• Plasmid pM220 (also known as pM210) was used as the source of the TPIl promoter fragment. E. coli RRI transformed with pM220 has been deposited with ATCC under accession number 39853. Plasmid pM220 was digested with Eco RI and the 0.9 kb fragment comprising the TPI1 promoter was isolated by agarose gel electrophoresis and the ends were blunted with DNA polymerase I (Klenow fragment). Kinased Xba I linkers were added to the fragment, which was then digested with Bgl II and Xba I.
• Plasmid pDR1107 was constructed by subcloning the 900 bp Bgl II-Eco RI TPI1 promoter fragment of pM220 into pIC7 (Marsh, Erfle and Wykes, Gene 32: 481-485, 1984) to generate plasmid pDRHOl. Plasmid pDRH01 was digested with Hind III and Sph I to isolate the 700 bp partial TPI1 promoter fragment.
• the plasmid was digested with Hind III and Eco RI and the 0.9 kb fragment was isolated and ligated to a synthetic linker constructed by annealing oligonucleotides ZC708 ( 5' AATTGCTCGAGT 3' ) and ZC709 ( 3' CGAGCTCAGATC 5' ).
• the linker addition eliminates the Eco RI site at the 3' terminus of the TPIl promoter fragment and adds Xhol and Xba I sites. This fragment was then joined to Hind Ill-Xba I cut pUC13.
• the resultant plasmid was designated pZV134 ( Figure 8).
• This plasmid was digested with Eco RI and the 1.7 kb fragment containing MF ⁇ 1 was isolated and ligated into Eco RI cut pUC13.
• the resultant plasmid, designated pl92, was cleaved with Eco RI and the resultant 1.7 kb MF ⁇ 1 fragment was isolated and digested with Mbo II.
• the 550 bp Mbo II-Eco RI fragment was isolated and ligated to kinased Sal I linkers. The linkered fragment was cut with Sal I.
• the resulting 0.3 kb Sal I fragment was ligated into Sal I cut pUC4 (Vieira and Messing, Gene.
• Plasmid pZV24 (Example 2) was digested with Sph I and Bgl II and the 0.8 kb ADHI promoter-BARl fragment was isolated. Plasmid p489 was cleaved with Bam HI and the 0.3 kb MF ⁇ 1 fragment was isolated. These two fragments were joined in a three part ligation to Sph I+Bam HI cut YEp13. The resultant plasmid was designated pZV69 ( Figure 11).
• Plasmid pZV71 was digested with Xba I and Pst I and the 1.07 kb fragment was isolated.
• the ADHI promoter was isolated as a 0.42 kb Sph I-Xba I fragment from pZV24. These two fragments were joined, in a three part ligation, to Sph I+Pst I cut pUC18.
• the resulting plasmid, pZV73 was digested with Sph I and Bam HI and the 1.5 kb fragment comprising the expression unit was isolated and ligated into the Sph I+Bam HI cut YEp13 to form pZV-75 ( Figure 10).
• the BARl-MF ⁇ 1 fusion units from pZV69 and pZV75 were subcloned with the TPIl promoter into pUC18 ( Figure 11).
• Plasmid pZV69 was digested with Eco RI and Bam HI and the 0.55 kb fragment containing the fusion was isolated.
• the 0.9 kb TPIl promoter fragment was isolated from pZV118 by digestion with Hind III and Eco RI.
• a three part ligation was done by using the .55 kb BARl-MF ⁇ 1 fragment, the 0.9 kb TPIl promoter fragment and pUC19 cut with Hind III and Bam HI.
• the resultant plasmid was designated pSW59.
• Plasmid pZV75 was digested with Eco RI and Bam HI to isolate the 954 bp BARl-MF ⁇ 1 fusion fragment. This BARl-MF ⁇ 1 fragment was ligated in a three part ligation with the 0.9 kb Hind III+Eco RI TPIl promoter fragment and pUC18 cut with Hind III and Bam HI to generate plasmid pSW60.
• Plasmid pJH66 was linearized with Eco RI and blunt-ended with Klenow fragment.
• Kinased Bam HI linkers 5' CCGGATCCGG 3' ) were added and excess linkers were removed by digestion with Bam HI before religation.
• the resultant plasmid, pSW8, was cut with Sal I and Bam HI to isolate the 824 bp fragment encoding amino acids 252 through 525 of BARl.
• This BARl fragment was fused to a fragment encoding the C-terminal portion of substance P (Munro and Pelham, EMBO J . , 2: 3087-3093, 1984).
• Plasmid pPM2 containing the synthetic oligonucleotide sequence encoding the dimer form of substance P in M13mp8, was obtained from Munro and Pelham. Plasmid pPM2 was linearized by digestion with Bam HI and Sal I and ligated with the 824 bp BARl fragment. The resultant plasmid pSWl4 was digested with Sal I and Sma I to isolate the 871 - bp BARl- substance P fragment. Plasmid pZV16 ( Figure 10) was cut with Xba I and Sal I to isolate the 767 bp 5' coding sequence of BARl.
• This fragment was ligated with the 871 bp BARl-substance P fragment in a three part ligation with pUC18 cut with Xba I and Sma I.
• the resultant plasmid was designated pSW15.
• Plasmid pSW15 was digested with Xba I and Sma I to isolate the 1.64 kb BARl-substance P fragment.
• the ADHI promoter was obtained from pRL029, comprising the 0.54 kb Sph I-Eco RI fragment containing the ADHI promoter and 116 bp of the BARl 5' coding region from pZV24 in pUC18.
• Plasmid pRL029 was digested with Sph I and Xba I. to isolate the 0.42 kb ADHI promoter fragment.
• the TPIl terminator (Alber and-Kawasaki, J. Mol. - Appl. Gen. 1. 419-434, 1982) was provided as a 0.7 kb Xba I+Eco RI fragment in pUC18.
• the linearized fragment containing the TPIl terminator and pUC18 with a Klenow filled Xba I end and an Sph I end was ligated with the 0.42 kb ADHI promoter fragment and the 1.64 kb BARl-substance P fragment in a three part ligation to produce plasmid pSW22.
• Plasmid pSW94 was then constructed ( Figure 13).
• the 2.3 kb fragment containing the BARl-substance P fusion and the TPIl terminator was isolated from plasmid pSW22 as an Xba I-Sst I fragment.
• the Hind Ill-Xba I TPIl promoter fragment isolated from pZV134 was joined to the BARl-substance P-TPI1 terminator fragment in a three part ligation with Hind III+SstI cut pUC18.
• the resultant plasmid, pSW81 was cleaved with Hind III and Eco RI to isolate the 1.02 kb fragment containing the TPIl promoter and the 5' 116 bp of BARl.
• Plasmid pSW60 was cut with Eco RI and Bam HI to isolate the 954 bp BARl-MF ⁇ 1 fusion fragment. Plasmid pSW81 was cut with Hind III and Eco RI to isolate the 1.02 kb TPIl promoter-BARl fragment which was joined with the BARl-MF ⁇ 1 fusion fragment in a three part ligation into Hind III+Bam HI cut YEp13. The resultant plasmid was designated pSW95.
• Plasmid pZV16 was digested with Eco RI and Sal I. The isolated 651 bp BARl fragment was ligated with a kinased Hind III-Eco RI BARl specific adaptor (produced by annealing oligonucleotides ZC566:
• Plasmid pM220 provided the TPIl promoter fused to the MF ⁇ l prepro sequence. Plasmid pM220 was digested with Bgl II and Hind III to isolate the 1.2 kb TPIl promoter-MF ⁇ 1 prepro fragment.
• the 3' portion of the BARl coding region was obtained by cutting pZV9 with Sal I and Bam HI to isolate the 1.3 kb BARl fragment.
• the 684 bp Hind Ill-Sal I BARl fragment, the 1.2 kb Bgl II-Hind III TPIl promoter-MF ⁇ 1 prepro fragment and the 1.3 kb Sal I-Bam HI BARl fragment were joined with YEp13 linearized with Bam HI in a four part ligation.
• the construct with the desired, orientation of promoter and MF ⁇ 1-BARl fusion was designated pZVlOO ( Figure 14).
• a 1 kb Hind III-Pst I fragment comprising the TPIl promoter and the truncated MF ⁇ l prepro sequence from pM220 was joined to the 0.82 kb MF ⁇ 1 prepro-BARl fragment isolated from pZV102 in a three part ligation with YEp13 cut with Hind III and Bam HI.
• the resultant plasmid was designated pZV105.
• Plasmid pZV105 was cleaved with Hind III to isolate the 1.2 kb TPIl promoter-MF ⁇ 1 prepro fragment.
• Plasmid pZV102 was digested with Hind III to isolate the vector fragment containing the terminal ⁇ -factor copy.
• Plasmid pSW61 was linearized by a partial digestion with Hind III. Plasmid pZV102 was digested with Hind III to isolate the 0.3 kb BARl-MF ⁇ 1 fragment. This fragment was ligated into the linearized pSW61.
• the plasmid with the insert in the correct orientation at the Hind III site 264 bp 3' to the MF ⁇ 1 start codon was designated pSW70.
• Plasmid pSW70 was cleaved with Eco RI and Bam HI to isolate the 361 bp BARl-MF ⁇ 1 fragment.
• Plasmid pSW81 ( Figure 13) was digested with Hind III and Eco RI to isolate the 1.02 kb TPIl promoter-BARl fragment. This fragment was joined to the BARl-MF ⁇ 1 fragment in a three part ligation with YEp13 linearized with Hind III and Bam HI.
• the resultant plasmid, pSW96 contains the TPIl promoter and 356 bp of the 5' coding sequence of BARl fused to one copy of the ⁇ -factor coding sequence.
• the second BARl-MF ⁇ 1 construct containing 767 bp of BARl fused to one copy of the MF ⁇ 1 coding sequence was made using pZV75 as the source of the BARl fragment ( Figure 17).
• Plasmid pZV75 was digested with Eco RI and Bam HI to isolate the 954 bp BARl-MF ⁇ 1 fragment.
• Plasmid pZV101, containing the MF ⁇ l prepro sequence fused to BARl. was cut with Pst I and Eco RI to isolate the 0.27 kb MF ⁇ 1 prepro-BARl fragment.
• This fragment was joined to the 954 bp BARl-MF ⁇ 1 fragment in a three part ligation with pUC13 linearized with Pst I and Bam HI.
• the resultant plasmid, pZV104 was cleaved with Hind III to isolate the 0.70 kb BARl-MF ⁇ 1 fragment.
• This fragment was ligated to pSW61 which was linearized by partial digestion with Hind III.
• the plasmid with the insert in the correct orientation at the Hind III site 264 bp 3' to the start codon of MF ⁇ 1 was designated pSW74. Plasmid pSW74 was cut with Eco RI and Bam HI to isolate the 738 bp BARl-MF ⁇ 1 fragment.
• Plasmid pSW81 was cut with Hind III and Eco RI to isolate the 1.02 kb TPIl promoter-BARl fragment. This fragment was joined to the 738 bp BARl-MF ⁇ 1 fragment in a three part ligation with Hind III+Bam HI cut YEp13.
• the resultant plasmid, pSW97 contains the TPIl promoter and 767 bp of the 5' end of BARl fused to the single copy of the ⁇ -factor coding sequence.
• Plasmid pSW73 comprises the TPIl promoter, MF ⁇ 1 signal peptide and prepro sequence and the coding region for the four copies of ⁇ -factor in YEp13.
• the transformants were spotted onto a lawn of
• Plasmid pSW67 comprising the TPIl promoter, MF ⁇ l signal peptide, prepro and the coding region for one copy of ⁇ -factor in YEpl3 was used as a control for plasmids pSW96 and pSW97.
• Plasmids pSW98 and pSW99 are YEp13-based plasmids comprising the S. cerevisiae TPIl promoter, a 355 bp or 767 bp fragment of the BARl gene, including the mutated signal peptide cleavage site, and one copy of the ⁇ -factor coding sequence.
• the signal peptide mutation was introduced by standard in vitro mutagenesis methods (Zoller et al., Manual for Advanced Techniques in Molecular Cloning Course, Cold Spring Harbor Laboratory, 1983) using a phage M13 template and a synthetic mutageni ⁇ oligonucleotide (sequence 5'ATTACTGCTCCTACAAACGAT3').
• the phage template pSW54 was constructed by ligating the 0.54 kb
• Sph I-Eco RI fragment of pSW22 with Sph I-Eco RI digested M13mp19 Following in vitro mutagenesis, potentially mutagenized plaques were screened by plaque hybridization wi.th 32 P-labeled mutagenic oligonucleotide and were sequenced to confirm the presence of the mutation.
• plasmid pSW82 was digested with Hind III and Eco RI and with Bgl II and Eco RI and the resulting 1.02- kb fragments were isolated.
• the Hind III-Eco RI fragment of pSW82 was ligated with the Eco RI-Bam HI fragment of pSW74 and Hind III+Bam HI digested YEpl3 to form pSW99.
• the Bgl II-Eco RI fragment of pSW82 was ligated with the 0.30 kb Eco RI-Bam HI fragment of pSW70 and Bam HI digested YEp13, to form pSW98.
• Plasmid pSW98 includes the TPIl promoter, 355 bp of the 5' end of the mutagenized BARl sequence and a single copy of the ⁇ -factor coding sequence. Plasmid pSW99 contains the identical expression unit except for having 767 bp of the mutagenized BARl sequence.

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• 1986
  • 1986-10-20 HU HU865248A patent/HU206897B/hu not_active IP Right Cessation
  • 1986-10-20 AU AU65432/86A patent/AU6543286A/en not_active Abandoned
  • 1986-10-20 EP EP86906638A patent/EP0243465A1/en not_active Withdrawn
  • 1986-10-20 UA UA4203156A patent/UA41863C2/uk unknown
  • 1986-10-20 JP JP61505713A patent/JP2523562B2/ja not_active Expired - Fee Related
  • 1986-10-20 WO PCT/US1986/002198 patent/WO1987002670A1/en active Application Filing
  • 1986-10-23 IE IE280486A patent/IE63822B1/en not_active IP Right Cessation
  • 1986-10-23 CA CA000521206A patent/CA1316133C/en not_active Expired - Fee Related
  • 1986-10-25 CN CN86107554A patent/CN1027179C/zh not_active Expired - Lifetime
  • 1986-10-27 CZ CS867764A patent/CZ284251B6/cs not_active IP Right Cessation
  • 1986-10-27 SK SK7764-86A patent/SK279041B6/sk unknown
• 1987
  • 1987-06-23 DK DK320287A patent/DK320287D0/da not_active Application Discontinuation
  • 1987-06-24 FI FI872801A patent/FI872801A/fi not_active Application Discontinuation
• 1991
  • 1991-04-02 AU AU74003/91A patent/AU676132B2/en not_active Ceased

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