WO2002018570A1 - Yeast transformant producing recombinant human parathyroid hormone and method for producing the hormone - Google Patents

Yeast transformant producing recombinant human parathyroid hormone and method for producing the hormone Download PDF

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WO2002018570A1
WO2002018570A1 PCT/KR2001/001447 KR0101447W WO0218570A1 WO 2002018570 A1 WO2002018570 A1 WO 2002018570A1 KR 0101447 W KR0101447 W KR 0101447W WO 0218570 A1 WO0218570 A1 WO 0218570A1
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hpth
yeast
yapsin
yps3
saccharomyces cerevisiae
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PCT/KR2001/001447
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English (en)
French (fr)
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Sang Ki Rhee
Hyun Ah Kana
Bong Hyun Chung
Su Min Ko
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Dong Kook Pharmaceutical Co., Ltd.
Korea Research Institute Of Bioscience And Biotechnology
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Priority to US10/363,329 priority Critical patent/US7244591B2/en
Priority to JP2002524073A priority patent/JP3957630B2/ja
Priority to EP01958618A priority patent/EP1313848B1/en
Priority to GB0303081A priority patent/GB2381273B/en
Priority to DE10196565T priority patent/DE10196565B4/de
Priority to AU2001280242A priority patent/AU2001280242A1/en
Publication of WO2002018570A1 publication Critical patent/WO2002018570A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • 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
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/94—Saccharomyces
    • Y10S435/942—Saccharomyces cerevisiae

Definitions

  • the present invention relates to the production of human parathyroid hormone (hereinafter referred to as "hPTH' ) . More particularly, the present invention relates to the use of Saccharomyces cerevisiae mutant strains which are genetically disrupted in at least one of the yapsin family of aspartic proteases YP ⁇ 1, YPS2 and YPS3 and transformed with an expression vector anchoring a hPTH gene, in producing the hormone.
  • hPTH' human parathyroid hormone
  • hPTH is a peptide consisting of 84 amino acid residues, produced by the parathyroid gland. hPTH maintains calcium homeostasis in the kidneys and bones, having the physiological function of promoting calcium metabolism and osteogenesis. In the U.S.A. and the Europe, estrogen or calcitonin have been predominantly used as therapeutics for osteoporosis, but are found to be obstructive of bone absorption. Accordingly, leading pharmaceutical companies of the world are intensively and extensively studying hPTH for the development of therapeutics for osteoporosis by taking advantage of the ability of hPTH to promote osteogenesis .
  • yeast a single cell eukaryote
  • yeast has the advantage of expressing and secreting properly folded- and thus active- proteins because it is very similar to higher species in the gene transcription and translation systems, and its protein- secretion system.
  • yeast is advantageous as a host for producing proteins of interest in that yeast secretes few extracellular proteins making it easy to recover and purify exogenous proteins.
  • the yeast Saccharomyces cerevisiae is a GRAS (generally recognized as safe) microorganism that is not pathogenic to the body and does not produce endotoxins. With the anticipation of being very useful as a producer of medicinal recombinant hPTH, Saccharomyces cerevisiae has been studied in developing hPTH expression systems.
  • Saccharomyces cerevisiae is not industrially utilized as such a host because the extracellularly secreted recombinant hPTH is degraded by endogenous proteolytic enzymes of the yeast' s own, so that only a small amount of the intact molecule of hPTH can be recovered (Gabrielsen et al., Gene 90, 255(1990) ) .
  • hPTH-related protein was directly ligated to the 3' -end of the yeast ubiquitin gene to produce a non- cleavable hPTH-related protein (Rian et al . , Eur. J. Biochem. , 213, 641 (1993)).
  • protein mutants are medicinally used, there are required stringent tests to obtain permission for their medicinal use, because they are generally recognized as new medicines.
  • gene fusion technology it is necessary to remove the fusion site by expected digestion because proteins of interest may be produced at relatively yields owing to the presence of the fusion site.
  • the present inventors developed a method in which hPTH cleavage can be prevented to a significant extent simply by adding L-arginine at high concentrations to the culture media (Chung and Park, Biotechnol . Bioeng. 57, 245
  • the present inventors made a YAP3 gene-disrupted yeast strain (yap3 ⁇ ) , and constructed an hPTH production system by use of the yeast mutant. Upon culturing in flasks, the hPTH production system was found to prevent the cleavage of hPTH at an efficiency of as high as 80 %, thus producing the intact molecule of hPTH at high yield (Kang et al . , Appl . Microbiol. Biotechnol., 50, 187 (1998); Korean Pat. No. 0246932, yielded Dec. 8, 1999) .
  • the present inventors created a yeast mutant in which both of the genes are disrupted (yap3 ⁇ /mkc7 ⁇ ) for use in the observation of the influence of the enzymes on the hPTH cleavage at the terminal culture stage.
  • yap3 ⁇ and yap3 ⁇ /mkc7 ⁇ Choi, et al . , J. Microbiol Biotechnol 9, 679 (1999)).
  • YPS3 was found to have 50 % homology with both of YPSl and YPS2, while 35 % and 25 % homology was found between YPS6 and BARl and between YPS7 and PEP4, respectively. From the homology between YPS3 and YPSl, the present inventors drew the deduction that the hPTH cleavage occurring at the terminal culture stage of the ypsl ⁇ (previous yap3 ⁇ ) strain might be performed by yapsin 3.
  • the present invention has an object of providing a protein expression system, which can produce hPTH at high yield. It is another object of the present invention to provide a method for producing hPTH at high yield.
  • hPTH posttranslational modification of hPTH allows modification and adaptation leading to the present invention.
  • the present inventors found that the disruption of yapsin genes results in a surprising decrease in the endogenous degradation of recombinant hPTH in Saccharomyces cerevisiae.
  • Saccharomyces cerevisiae mutant strain in which both of YPSl and YPS3 genes or all of YPSl, YPS2 and YPS3 genes are disrupted.
  • Fig. 1 is a diagram showing a process of constructing a cassette for disrupting the YPS3 gene by use of a pop-out URA3 selection marker;
  • Fig. 2 is a diagram showing a process of disrupting the YPS3 gene of Saccharomyces cerevisiae and recovering the URA3 selection marker;
  • Fig. 3 shows SDS-PAGE results of hPTH molecules obtained from the cultures of Saccharomyces cerevisiae 2805 (lane 1), Saccharomyces cerevisiae 2805/pGlO-hPTHl (lanes 2 and 3) , Saccharomyces cerevisiae SY28Y3/pG10-hPTHl (lanes 4 and 5), and Saccharomyces cerevisiae SY28Y4/pG10-hPTHl (lanes 6 and 7), along with 1 ⁇ g of native hPTH (C) and a prestained protein molecular weight marker (M) grown for 24 hours (a) and 48 hours (b) , wherein band i stands for intact hPTH (1-84 a.a.), band dl for a truncated hPTH (27-84 a.a.), and band d2 for hPTH (1-80 a.a.);
  • Fig. 4 shows SDS-PAGE results of hPTH molecules obtained from the cultures of Saccharomyces cerevisiae L3262 (lane 1) , Saccharomyces cerevisiae L3262/pGl0-hPTHl (lanes 2 and 3) , Saccharomyces cerevisiae SLH15/pG10-hPTHl (lanes 4 and 5) , Saccharomyces cerevisiae SLH16/pG10-hPTHl (lanes 6 and 7), Saccharomyces cerevisiae SLH17/pG10-hPTHl (lanes 9 and 9), and Saccharomyces cerevisiae SLH18/pG10-hPTHl (lanes 10 and 11), along with 1 ⁇ g of native hPTH (C) and a prestained protein molecular weight marker (M) grown for 24 hours (a) and 48 hours (b) , wherein band i stands for intact hPTH (1-84
  • Fig. 5 shows SDS-PAGE results of hPTH molecules obtained from the cultures of Saccharomyces cerevisiae L3262 (lane 1), YPSl/YPS3-double disruptants of Saccharomyces cerevisiae L3262 (a) : Saccharomyces cerevisiae L3262/pG10- hPTHl (lanes 2 to 7) and Saccharomyces cerevisiae SLHll/pGlO-hPTHl (lanes 8 to 13), YPSl/YPS2/YPS3-triple disruptants (b) of Saccharomyces cerevisiae L3262: Saccharomyces cerevisiae SLH16/pG10-hPTHl (lanes 2 to 7) and Saccharomyces cerevisiae SLH18/pG10-hPTHl (lanes 8 to 13) , along with 1 ⁇ g of native hPTH (C) and a prestained protein molecular
  • the present invention pertains to a method for producing recombinant hPTH from a yeast host which is unable to produce at least one of the yapsin family of proteases YPSl, YPS2 and YPS3.
  • the present invention comprises a method for producing the intact molecule of hPTH at a high yield by using as a host cell a yeast mutant which is unable to express at least one of the three proteases YPSl, YPS2 and YPS3.
  • YPSl and YPS2 exert their enzymatic activity mostly at the early culture stage of transformants for producing hPTH, while YPS3 cleaves hPTH mainly at the late culture stage.
  • hPTH can be obtained at a high yield in the early culture stage because of the lacking of YPSl, but at a poor yield in the late culture stage owing to the inevitable proteolytic activity of YPS3.
  • a mutant defective preferably in both YPSl and YPS3 (ypsl ⁇ /yps3 ⁇ ) , and more preferably in all of YPSl, YPS2 and YPS3 (ypsl ⁇ /yps2 ⁇ /yps3 ⁇ ) is used as the host.
  • Deletion of the proteases can be carried out by disrupting at least one gene selected from the group consisting of YPSl, YPS2 and YPS3 by use of an enzyme selection marker.
  • the yeast selection marker is not particularly limited, but is preferably a one that can pop-out.
  • a URA3 selection marker is provided in a form of a cassette.
  • the pop-out cassette containing a yeast selection marker has gene fragments coding for N- and C-terminal sites of YPSl, YPS2 and YPS3 at its both ends such that the target genes contained in the genome of yeast, i.e., ypsl, yps2 and yps3, can be disrupted by a homology recombination method. Any selection marker may be used if it is able to select the yeast which harbors the cassette in its genome.
  • the mutant yps3 ⁇ in which the yps3 gene is disrupted by a URA3 selection marker is denoted by yps3::URA3.
  • an hPTH gene is carried into yeast by an expression vector.
  • the present invention pertains to a recombinant expression vector to which an hPTH gene is inserted.
  • the expression vector useful in the present invention has means for expressing a gene in yeast and means for controlling the expression. Vector selection and recombinant vector construction are obvious to those who are skilled in the art and details thereof will be described in the following examples .
  • the present invention pertains to a transfor ant prepared from the ypsl ⁇ /yps3 ⁇ or ypsl ⁇ /yps2 ⁇ /yps3 ⁇ yeast mutant with the recombinant expression vector.
  • the recombinant vector pGlO-hPTHl is transformed into Saccharomyces cerevisiae mutants (ypsl ⁇ /yps3 ⁇ and ypsl ⁇ /yps2 ⁇ /yps3 ⁇ ) to create transformants SLHl6/pG10-hPTH and SLH18/pG10-hPTH, which were deposited in the Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology (KRIBB) with accession Nos. KCTC 0815BP and KCTC 0816BP, respectively, on Jul . 6, 2000.
  • KRIBB Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology
  • hPTH for the expression of hPTH in Saccharomyces cerevisiae, there was employed the plasmid pGlO-hPTHl, which contains an hPTH expression cassette composed of GAL10 promoter : :ppL: :hPTHdb?: :GAL7 terminator (Chung and Park, Biotechnol. Bioeng. 57, 245 (1998)).
  • a pop-out URA3 selection marker (URA3::tc5) cassette was prepared from a 1.8kb BamHI fragment derived from pTcUR3 (Kang et al., Appl . Microbiol. Biotechnol., 53, 575-582 (2000)).
  • Saccharomyces cerevisiae 2805 and Saccharomyces cerevisiae L3262a were used as parental cells for preparing YPS3-deleted mutants (Kang et al., J. Microbiol. Biotechnol., 8, 42-48 (1998)).
  • the cells used in the present invention were the yeast strains SLH11 (Kang et al . , Appl. Micribiol. Biotechnol., 59, 187 (1998)), SLH12 and SLH14 (Choi et al . , J. Biosci .
  • YPDG media yeast extract 1 %, Bacto peptone 2 %, glucose 1 %, galactose 1%) .
  • EXAMPLE 1 Establishment of YPS3 Gene-Disrupted Yeast Mutant
  • a recombinant vector containing a URA3 cassette for use in disrupting the YPS3 gene is illustrated in Fig. 1.
  • N- and C-terminal fragments of YPS3 gene necessary for the homology recombination leading to the disruption of the YPS3 gene were produced by PCR (polymerase chain reaction) using two pairs of primers, which were synthesized on the basis of the YPS3 base sequence of Saccharomyces cerevisiae, registered in GenBank.
  • PCR polymerase chain reaction
  • primers which were synthesized on the basis of the YPS3 base sequence of Saccharomyces cerevisiae, registered in GenBank.
  • a set of primers (5 1 - GACGAATTCCAGAAACGTCTGAGTGGAG-3 ' and 5 ' -
  • GCAGGATCCGTACTCTACCGAATGCCG-3 ' for amplifying the N- terminal fragment were designed to have recognition sites of EcoRI and BamEI at their respective 5' -terminal sites (underlined parts) while restriction sites of BamRI and Xbal were introduced into 5' -terminal sites of a set of primers ( 5 ' -CGCGGATCCCTATGCAGACCAGTGTGG-3 ' and 5 ' - .
  • CGCTCTAGACTGCATGCAAGGTCTGAC-3 ' for the amplification of the C-terminal fragment (underlined parts) .
  • the PCR was carried out in a thermal cycler, such as that manufactured by Perkin Elmer, identified as "GeneAmp PCR 2400", with 25 thermal cycles, each consisting of 95 °C/30 sec for denaturing, 55 °C/30 sec for annealing, and 72 °C/30 sec for extending, so as to produce a 800 bp and a 700 bp-DNA fragment encoding an N-terminal and a C-terminal region of the YPS3 gene region, respectively, from the genomic DNA of Saccharomyces cerevisiae.
  • the PCR products that is, the YPS3 N-terminal and C-terminal fragments, were double digested with restriction enzymes EcoRI/BamHI and BamHI/Xbal, respectively, followed by ligating the two restriction enzyme digests together into the pBluescript II KS(+) vector (Stratagen) which was previously treated with EcoRI/Xbal.
  • a URA3 pop- out selection marker was introduced, so as to construct the pB-yps3: :URA3 : tc5 vector for use in disrupting the YPS3 gene.
  • the YPS3 gene of Saccharomyces cerevisiae was disrupted by use of the URA3 cassettes, followed by recovering the URA3 selection marker therefrom, as illustrated in Fig. 2.
  • PCR was conducted to identify whether the Ura + transformants were disrupted in the YPS3 gene, after which the yps3 : :URA3 : tc5 transformants thus selected were spread onto 5-FOA plates to select the yps3::tc clones resulting from the pop-out of the URA 3 gene through homology recombination.
  • EXAMPLE 2 Establishment of Recombinant Yeast Strain Expressive of hPTH and Analysis of hPTH Expression
  • Saccharomyces cerevisiae wild-type strain 2805 and L3262, and Saccharomyces cerevisiae mutants S28Y4, SLH15 (yps3 ⁇ ) , SLH16 (ypsl ⁇ /yps3 ⁇ ) , SLH17 (yps2 ⁇ /yps3 ⁇ ) , and SLH18 (ypsl ⁇ /yps2 ⁇ /yps3 ⁇ ) were transformed with the expression vector pGlO-hPTHl, followed by the selection of Ura+ transformants.
  • These recombinant yeast strains capable of hPTH expression were subjected to hPTH expression analysis.
  • the yeast mutants were pre-cultured at 30 °C for 24 hours in minimal selective broths (amino acid-deficient yeast nitrogen substrate 0.67 %, glucose 2 %, casamino acid 0.5 %) .
  • Each of the cultures was inoculated in an amount of 2 % to a YPDG medium (yeast extract 1 %, bacto peptone 2 %, glucose 1 %, galactose 1 %) and then incubated at 30 °C for 48 hours. During this incubation, samples were withdrawn at 24 hours and 48 hours. 500 ⁇ l of each sample was centrifuged at 5,000 rpm for 5 min to separate a supernatant from biomass.
  • DOC deoxycholic acid
  • TCA trichloroacetic acid
  • the recombinant hPTH secreted from the Saccharomyces cerevisiae strains are separated on SDS-PAGE, appearing as three bands: i for the intact molecule (1-84); dl for an N-terminus truncated molecule (27-84); and d2 for a C-terminus truncated molecule (1-80) .
  • the proteins located in the i band and dl band were transferred onto a PVDF (polyvinylidene difluoride) membrane.
  • N-terminal amino acid sequencing of the proteins with the aid of Milligen/Biosearch M 600 protein sequencer identified the amino acid sequences Ser-Val-Ser-Glu-Ile and Lys-Leu-Gln- Asp-Val at the N-terminal regions of the proteins of the i and dl bands, respectively, indicating that the protein of the i band is the intact molecule of hPTH while the protein of the dl band is a truncated form (27-84) which is lacking in 26 amino acid residues of the N-terminal region of hPTH.
  • the d2 band its protein, though showing an electrophoretic band of 14 kDa larger than the intact molecule, was found to be a truncated hPTH form lacking 4 to 5 amino acid residues of the C-terminus (1-79, 80) as analyzed by HPLC, MALDI mass spectrometry, and C-terminal amino acid sequencing.
  • the characteristic electrophoretic pattern is reported to be attributed to the conformational change of the protein resulting from the removal of the C- terminus (Vad et al., Protein Expr. Purif. 13:396-402 (1998) ) .
  • hPTH molecules secreted from the non-transformed, wild-type Saccharomyces cerevisiae 2805, the transformed wild-type Saccharomyces 2805/pGlO-hPTHl, the transformed ypsl ⁇ mutant S28Y3/pG10-hPTHl, and the transformed yps3 ⁇ mutant S28Y4/pG10-hPTHl were subjected to SDS-PAGE electrophoresis.
  • the electrophoresis results are shown in Fig. 3.
  • the results, taken together and shown in Fig. 3 demonstrate that the main protease causative of the cleavage of hPTH in the early culture stage is Yapsin 1 and other proteases than Yapsin 1 are responsible for the cleavage of hPTH in the late culture stage.
  • the strain that is disrupted at the YPS3 gene only no significant effects can be obtained because the activity of Yapsin 1 already cleaves the hPTH to a significant extent in the early culture stage.
  • hPTH was expressed in the non-transformed wild- type Saccharomyces cerevisiae L3262 serving as a control, the transformed L3262/pG10-hPTHl, the transformed yps3 ⁇ mutant SLH15/pG10-hPTHl, the transformed ypsl ⁇ /yps3 ⁇ mutant SLH16/pG10-hPTHl, the transformed yps2 ⁇ /yps3 ⁇ mutant SLH17/pG10-hPTHl, and transformed ypsl ⁇ /yps2 ⁇ /yps2 ⁇ mutant SLHl8/pG10-hPTHl .
  • Secreted proteins were electrophoresed in the same manner as above and the electrophoresis results are shown for the sample withdrawn after 24 hours of incubation in
  • hPTH was expressed in the non- transformed wild-type Saccharomyces cerevisiae L3262 serving as a control, the transformed L3262/pG10-hPTHl, the transformed ypsl ⁇ mutant SLHll/pGlO-hPTHl, the transformed ypsl ⁇ /yps3 ⁇ mutant SLHl6/pG10-hPTHl, and transformed ypsl ⁇ /yps2 ⁇ /yps2 ⁇ mutant SLHl8/pG10-hPTHl .
  • yeast mutants which are disrupted both in Yapsin 1 (previously YPA3) and Yapsin 3 genes (y ⁇ sl ⁇ /yps3 ⁇ ) or all in Yapsi.nl, Yapsin 2 (previously MKC7) and Yapsin3 genes (ypsl ⁇ /yps2 ⁇ /yps3 ⁇ ) can secrete intact hPTH, useful as therapeutics for various disorders, at high yield by virtue of their inability to degrade the hormonal peptide.

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PCT/KR2001/001447 2000-08-31 2001-08-27 Yeast transformant producing recombinant human parathyroid hormone and method for producing the hormone WO2002018570A1 (en)

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Application Number Priority Date Filing Date Title
US10/363,329 US7244591B2 (en) 2000-08-31 2001-08-27 Yeast transformant producing recombinant human parathyroid hormone and method for producing the hormone
JP2002524073A JP3957630B2 (ja) 2000-08-31 2001-08-27 組換えヒト副甲状腺ホルモンを生産する形質転換酵母及び該ホルモンの生産方法
EP01958618A EP1313848B1 (en) 2000-08-31 2001-08-27 Yeast transformant producing recombinant human parathyroid hormone and method for producing the hormone
GB0303081A GB2381273B (en) 2000-08-31 2001-08-27 Yeast transformant producing recombinant human parathyroid hormone and method for producing the hormone
DE10196565T DE10196565B4 (de) 2000-08-31 2001-08-27 Rekombinantes Human-Parathyroidhormon erzeugender Hefe-Transformant und Verfahren zur Herstellung des Hormons
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100386836B1 (ko) * 2000-08-31 2003-06-09 동국제약 주식회사 재조합 인체 부갑상선 호르몬을 생산하는 형질전환효모 및재조합 인체 부갑상선 호르몬의 생산방법
WO2013098651A1 (en) * 2011-12-30 2013-07-04 Oxyrane Uk Limited Methods and materials for reducing degradation of recombinant proteins
US9206408B2 (en) 2007-04-03 2015-12-08 Oxyrane Uk Limited Microorganisms genetically engineered to have modified N-glycosylation activity
US9249399B2 (en) 2012-03-15 2016-02-02 Oxyrane Uk Limited Methods and materials for treatment of pompe's disease
US9347050B2 (en) 2010-09-29 2016-05-24 Oxyrane Uk Limited Mannosidases capable of uncapping mannose-1-phospho-6-mannose linkages and demannosylating phosphorylated N-glycans and methods of facilitating mammalian cellular uptake of glycoproteins
US9598682B2 (en) 2009-09-29 2017-03-21 Vib Vzw Hydrolysis of mannose-1-phospho-6-mannose linkage to phospho-6-mannose
US9689015B2 (en) 2010-09-29 2017-06-27 Oxyrane Uk Limited De-mannosylation of phosphorylated N-glycans
US10287557B2 (en) 2009-11-19 2019-05-14 Oxyrane Uk Limited Yeast strains producing mammalian-like complex N-glycans

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100470978B1 (ko) * 2003-01-02 2005-03-10 한국생명공학연구원 앱신 다중 결손 효모 변이 균주 및 이를 이용한 재조합 단백질의 생산 방법
CN1838968A (zh) 2003-08-08 2006-09-27 艾伯吉尼斯公司 针对甲状旁腺激素(pth)之抗体和其用途
US7318925B2 (en) * 2003-08-08 2008-01-15 Amgen Fremont, Inc. Methods of use for antibodies against parathyroid hormone
DE602006018634D1 (de) * 2005-08-03 2011-01-13 Asahi Glass Co Ltd Transformierte Hefezellen UND VERFAHREN ZUR HERSTELLUNG VON FREMDPROTEIN
WO2023215221A2 (en) * 2022-05-02 2023-11-09 North Carolina State University Engineered microorganisms with enhanced protein expression and secretion

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5420242A (en) * 1986-10-22 1995-05-30 Kaare M. Gautvik Production of human parathyroid hormone from microorganisms
WO1999037758A2 (en) * 1998-01-27 1999-07-29 The Board Of Regents Of The University And Community College System Of Nevada On Behalf Of The University Of Nevada-Reno Expression of proteolytically-sensitive peptides

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3752375T8 (de) * 1986-10-22 2005-03-10 Nps Allelix Corp., Mississauga Herstellung von humanem Parathormon
KR100246932B1 (ko) * 1997-09-12 2000-03-15 박호군 Yap3 유전자가 결손된 신규한 효모균주 및 그를 이용한 재조합 인체 부갑상선호르몬의 생산
ES2258371B1 (es) * 2000-08-31 2007-05-16 Dong Kook Pharmaceutical Co. Ltd. Levadura transformante productora de hormona paratiroidea humana y procedimiento para producir la hormona.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5420242A (en) * 1986-10-22 1995-05-30 Kaare M. Gautvik Production of human parathyroid hormone from microorganisms
WO1999037758A2 (en) * 1998-01-27 1999-07-29 The Board Of Regents Of The University And Community College System Of Nevada On Behalf Of The University Of Nevada-Reno Expression of proteolytically-sensitive peptides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
APPL. MICROBIOL. BIOTECHNOL., vol. 50, no. 2, 1998, pages 187 - 192 *
DATABASE CA [online] KARLSRUHE (DE); KANG H.A. ET AL.: "Efficient production of intact human parathyroid hormone in a saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3)", XP002962057, accession no. STN Database accession no. 130:955 *
DATABASE CA [online] KARLSRUHE (DE); SONG ET AL.: "Overproduction of human parathyroid hormone by fed-batch culture of a saccharomyces cerevisiae mutant lacking yeast aspartic protease 3", XP002962058, accession no. STN Database accession no. 132:63199 *
PROCESS BIOCHEM., vol. 35, no. 5, 2000, OXFORD, pages 503 - 508 *

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KR100386836B1 (ko) * 2000-08-31 2003-06-09 동국제약 주식회사 재조합 인체 부갑상선 호르몬을 생산하는 형질전환효모 및재조합 인체 부갑상선 호르몬의 생산방법
US10023854B2 (en) 2007-04-03 2018-07-17 Oxyrane Uk Limited Microorganisms genetically engineered to have modified N-glycosylation activity
US9206408B2 (en) 2007-04-03 2015-12-08 Oxyrane Uk Limited Microorganisms genetically engineered to have modified N-glycosylation activity
US9222083B2 (en) 2007-04-03 2015-12-29 Oxyrane Uk Limited Microorganisms genetically engineered to have modified N-glycosylation activity
US10392609B2 (en) 2009-09-29 2019-08-27 Oxyrane Uk Limited Hydrolysis of mannose-1-phospho-6-mannose linkage to phospho-6-mannose
US9598682B2 (en) 2009-09-29 2017-03-21 Vib Vzw Hydrolysis of mannose-1-phospho-6-mannose linkage to phospho-6-mannose
US11225646B2 (en) 2009-11-19 2022-01-18 Oxyrane Uk Limited Yeast strains producing mammalian-like complex n-glycans
US10287557B2 (en) 2009-11-19 2019-05-14 Oxyrane Uk Limited Yeast strains producing mammalian-like complex N-glycans
US10011857B2 (en) 2010-09-29 2018-07-03 Oxyrane Uk Limited Mannosidases capable of uncapping mannose-1-phospho-6-mannose linkages and demannosylating phosphorylated N-glycans and methods of facilitating mammalian cellular uptake of glycoproteins
US9689015B2 (en) 2010-09-29 2017-06-27 Oxyrane Uk Limited De-mannosylation of phosphorylated N-glycans
US10344310B2 (en) 2010-09-29 2019-07-09 Oxyrane Uk Limited De-mannosylation of phosphorylated N-glycans
US9347050B2 (en) 2010-09-29 2016-05-24 Oxyrane Uk Limited Mannosidases capable of uncapping mannose-1-phospho-6-mannose linkages and demannosylating phosphorylated N-glycans and methods of facilitating mammalian cellular uptake of glycoproteins
WO2013098651A1 (en) * 2011-12-30 2013-07-04 Oxyrane Uk Limited Methods and materials for reducing degradation of recombinant proteins
US10648044B2 (en) 2012-03-15 2020-05-12 Oxyrane Uk Limited Methods and materials for treatment of Pompe's disease
US9249399B2 (en) 2012-03-15 2016-02-02 Oxyrane Uk Limited Methods and materials for treatment of pompe's disease

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EP1313848A1 (en) 2003-05-28
KR100386836B1 (ko) 2003-06-09
US20040029252A1 (en) 2004-02-12
AU2001280242A1 (en) 2002-03-13
US7244591B2 (en) 2007-07-17
ES2258371B1 (es) 2007-05-16
DE10196565B4 (de) 2007-02-01
DE10196565T1 (de) 2003-07-03
KR20020017754A (ko) 2002-03-07
GB0303081D0 (en) 2003-03-19
GB2381273B (en) 2004-12-22
GB2381273A (en) 2003-04-30
EP1313848B1 (en) 2006-07-05
JP2004507270A (ja) 2004-03-11
CN1449441A (zh) 2003-10-15
ES2258371A1 (es) 2006-08-16
JP3957630B2 (ja) 2007-08-15

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