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WO2013191369A1 - Souche de levure au gène hpgas1 interrompu et procédé de production d'une protéine recombinante à l'aide de celle-ci - Google Patents

Souche de levure au gène hpgas1 interrompu et procédé de production d'une protéine recombinante à l'aide de celle-ci Download PDF

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WO2013191369A1
WO2013191369A1 PCT/KR2013/003581 KR2013003581W WO2013191369A1 WO 2013191369 A1 WO2013191369 A1 WO 2013191369A1 KR 2013003581 W KR2013003581 W KR 2013003581W WO 2013191369 A1 WO2013191369 A1 WO 2013191369A1
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gene
strain
hpgasl
polymorpha
protein
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PCT/KR2013/003581
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Korean (ko)
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권오석
황동현
오두병
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한국생명공학연구원
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Priority claimed from KR1020130045467A external-priority patent/KR101498012B1/ko
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • Hp GA Gene Fragmenting Yeast Strain and Method of Production of Recombinant Protein Using the Same
  • the present invention relates to a mutant strain that breaks a novel gene to improve protein secretion ability and a foreign recombinant protein production method using the mutant strain.
  • Protein drug development began in the 1970s with the introduction of genetic recombination technology.
  • the recombinant protein pharmaceutical production technology which produces a large amount of useful proteins such as hormones, antibodies, vaccines, and biofunctional proteins in a microbial host cell and develops them into pharmaceuticals, utilizes representative microorganisms that utilize microorganisms as cell factories.
  • the demand for high-purity proteinaceous medicines for the treatment of intractable diseases is growing exponentially, and recombinant medicines using microbial expression systems capable of mass production at low cost Production technology development is expected to contribute greatly to the growth of the future pharmaceutical industry.
  • Early protein drugs were sometimes isolated from human tissue or blood, but due to serious problems such as viral infections, including HIV and hepatitis, and the potential for residual cancer-causing agents,
  • Recombinant protein production technology is a technology that produces high value-added medical proteins with limited production cost by producing genes derived from higher organisms from a variety of microorganisms through gene recombination technology developed relatively).
  • the demand for high-purity proteinaceous drugs is expected to soar due to the increase of diseases and the improvement of national medical standards. Therefore, there is a need for a technology development research capable of mass production of new functional recombinant protein at a relatively low cost using various microorganisms that are harmless to the human body.
  • methanol magnetization yeast Hansenula polymorphaV (3 ⁇ 4 £ / / 3 polymorphaV): It is possible to cultivate relatively high concentrations easily using methanol, a cheap raw material as a carbon source, and a strong promoter derived from several genes involved in metabolism. In addition, the foreign gene is transferred to the host cell's chromosomal DNA. Multicopy It can be integrated) even during high concentration cultivation : it has the advantage of keeping stable.
  • the present inventors have proposed a novel beta-1 that is critical for cell wall composition and permeability determination in order to further increase the production of protein secretion of the existing Hansenula polymorpho ( ⁇ e / / a polyworpha) strain, which is advantageous for the production of foreign recombinant proteins.
  • HpGASl gene having 3-glucanosyl transglycosy lase (beta-1, 3-glucanosyl transglycosy lase) activity was secured, it was confirmed that the foreign recombinant protein secretion ability increases when the gene is deleted, through the present invention
  • the present invention was completed by revealing that a single-deleted Hansenula polymorpha mutant strain having a single deletion of HpGASl gene can be usefully used as a resource for secretory production of a pharmaceutical recombinant protein.
  • Another object of the present invention to provide a method for producing a yeast strain with improved protein secretion ability.
  • Another object of the present invention is to provide a foreign recombinant protein production method.
  • Another object of the present invention is to provide a use of a Hanshenula polymorpho Ofe ⁇ ey / polymorpha) strain that is deficient in the HpGASl gene as set forth in SEQ ID NO: 1 to enhance protein secretion ability.
  • the present invention is HpGASl described in SEQ ID NO: 1 It provides a Hanshenula polymorpha 03 ⁇ 4 7se // a polyworpha) variant strain of Accession No. KCTC12220BP with improved gene secretion ability.
  • the present invention also provides a method for producing a yeast strain with improved protein secretion ability.
  • the present invention also provides a foreign recombinant protein production method.
  • the present invention provides a use of a Hansennura polymorpha 03 ⁇ 472s 3 ⁇ 4 // a poly orpha) mutant strain lacking the HpGASl gene as set forth in SEQ ID NO: 1 for improving protein secretion ability.
  • Figure 2 shows the results of searching for homology to the sequence of the HpGAS2 gene described in SEQ ID NO: 2 using BLAST of NCBI.
  • Hydrophobic region Hydrophobic region
  • Figure 4 is a diagram showing the manufacturing process of the ⁇ gene disruption cassette.
  • FIG. 5 is a diagram illustrating a manufacturing process of the HpGASl gene disruption cassette.
  • FIG. 6 is a diagram showing the manufacturing process of the ⁇ 5 gene disruption cassette.
  • FIG. 8 is a photograph showing the cell morphological features of HpGASl and HpGAS2 single deletion mutants.
  • A 2% glucose complex medium (37 0 C); B: 2% glucose complex medium (42 ° C.);
  • Hanshenula polymorpha strain (polymorpha DLl AHpGASl ⁇ 3 ⁇ 43 ⁇ 45 / pDLGUK-HpGASl) lacking the HpGASl gene transformed with HpGASl expression vector (pDLGUK-HpGASl);
  • Hansenula polymorpha strain lacking the HpGASl gene transformed with HpGAS2 expression vector P DLGUK-HpGAS2 (.polymorpha DLl AHpGASl ⁇ j pDLGUK-HpGAS2);
  • Hansenula polymorpha DL1 wild type strain (.polymorpha DLl);
  • FIG. 11 is a photograph comparing Glucose oxidase (GOD) secretion in HpGASl and HpGAS2 single deletion strains:
  • HpGAS-deficient GOD producing strain [polymorpha AffpGAS pOLUOI-GODiR)].
  • Figure 13 is a photograph comparing human serum albumin (HSA) secretion in HpGASl and HpGAS2 single deletion strains:
  • HSA producing strains deficient in HpGAS (.polymorpha G3T8 AHpGAS2).
  • 14 is a diagram showing a process of H PGASl gene disrupted strains and H P UR / deletion. '
  • Figure 15 is a photograph confirming the physiological characteristics of strains transformed HpGASl and 3 ⁇ 4? Fi45 gene expression vector in 5. cerevisiae BY4741 lacking the ScGASl gene:
  • A 2% glucose complex medium
  • B Sodium Dodecyl Sulfate (SDS) ⁇ 2% galactose and 1% raffinose complex medium
  • C Congo Red (CR)-2% galactose and 1% raffinose minimal medium
  • Saccharomyces cerevisiae strain (5. cerevisiae BY4741 15c ⁇ 5i / YEp352ScGAPDHpt -HpGAS2) that lacks the 5 45? Gene transformed with ⁇ A expression vector (YEp352ScGAPDHpt-HpGAS2); and
  • Saccharomyces cerevisiae strain S. cerevisiae BY4741 l & i3 ⁇ 45i / YEp352ScGAPDHpt
  • Saccharomyces cerevisiae strain S. cerevisiae BY4741 l & i3 ⁇ 45i / YEp352ScGAPDHpt
  • YEp352ScGAPDHpt empty site
  • the present invention provides Hansenula polymorpha ( ⁇ ;? se // a polymorpha) mutant strain of Accession No. KCTC12220BP with improved protein secretion ability, wherein the HpGASl gene described in SEQ ID NO: 1 is deleted.
  • the inventors obtained HpGAS gene candidates for translating proteins having amino acid sequences similar to those of other yeast Gasl proteins in the Hanshenula polymorpha DL1 genomic information database.
  • two Hansenula polymorpha genes ( ⁇ ⁇ 5 ⁇ HpGAS2) o] were analyzed to translate Gasl proteins of other yeasts and proteins having high amino acid sequence homology (see FIGS. 1 and 2).
  • HpGASl gene of a novel sequence that translates a protein having higher homology with the amino acid sequence of ScGasl protein of Saccharomyces cerevisiae The amino acid sequence and the active domain conserved in several Gasl proteins exist in the amino acid sequence of the translating protein It was confirmed that (see Figure 3).
  • HpGASl ⁇ ⁇ ⁇ 5 gene was disrupted first, the Hansenula polymorpha U A3 gene was disrupted with a disruption cassette, followed by homologous recombination using HpGASl and H P GAS2 gene disruption cassettes. The HpGASl and H P GAS2 genes of the morpha strains were respectively disrupted.
  • the ⁇ gene disruption cassette was prepared using an N-terminal fragment of the HpURA3 gene, a C-terminal fragment of the HpURA3 gene, and a Zeocin resistance gene (Zeo R ) cassette. It is preferably a crushed cassette, but is not limited thereto.
  • the HpGASl gene disruption cassette comprises an N-terminal fragment of the HpGASl gene, a C-terminal fragment of the HpGASl gene, and 7ac -y3 ⁇ 4? N-terminal fragment of cassette, 1 acZ ⁇ HpURA3- 1 acZ? It is preferable to use a shredding cassette produced using a sheet C-terminal fragment, but is not limited thereto.
  • the inventors of the present invention in order to disrupt the ⁇ 3 gene to distinguish gene disruption, the N terminal segment of the H P URA3 gene, the C terminal segment of the HpUM3 gene and Zeocin resistance gene (Zeo R ) cassette Using the «fragment cassette was transformed to Hanshenula polymorpha strain and then the corresponding gene on the chromosome was disrupted by homologous recombination method (see Fig. 4).
  • the N-terminal fragment of the HpGASl gene, the C-terminal fragment of the HpGASl gene, lacZ-HpUM3-lacZ 7 ⁇ hent ⁇ N-terminus fragment, and 1 acZ-HpURA3- 1 acZ? ⁇ ⁇ terminal fragment were prepared in the fragmented strain.
  • One disrupted cassette was transformed to prepare a strain in which the HpGASl gene was crushed (see FIG. 5), and it was confirmed that the protein secretion ability of the strain in which the ⁇ i S gene was crushed was improved.
  • the recombinant yeast strain is Saccharomyces (5ac 3 ⁇ 4 / 3 ⁇ 4z ⁇ es), Kluberomyces Uduyveroinyces, Picky ⁇ iPichia, Hanshenula 03 ⁇ 4 / 7se //) or It is preferably any one selected from the group consisting of Candida genus, and more preferably, but not limited to Hanshenula polymorpha strain.
  • the foreign recombinant production method preferably further comprises the step of culturing the mutant strain using a bioreactor, but is not limited thereto.
  • the inventors have introduced a vector comprising a gene encoding glucose oxidase (GOD) or human serum albumin (HSA) to introduce HpGASl or GOD-producing strains and HSA-producing strains that produce foreign recombinant proteins.
  • GOD glucose oxidase
  • HSA human serum albumin
  • the Hanshenula polymorpha strain in which the HpGASl gene of the present invention is singly disrupted can be usefully used as a resource for the production of foreign recombinant proteins.
  • the present invention provides a use of a Hansennura polymorpha (? / 7 «? / 3 polymorpha) variant strain that lacks the HpGASl gene as set forth in SEQ ID NO: 1 to improve protein secretion ability.
  • HpGASl genes were identified through translation and search.
  • HpGAS gene candidates were obtained through a tblastn program for translating proteins having amino acid sequences similar to those of Gasl proteins.
  • the two highly homologous genes were compared and selected for homology between the other yeast Gasl protein and amino acid sequence and named HpGASl gene (SEQ ID NO: 1) and ⁇ ⁇ 45 gene (SEQ ID NO: 2) (Table 1).
  • the present inventors cloned the Zeocin resistance gene (Zeo R ) between URA3 gene segments with the URA3 disruption cassette to disrupt the ⁇ 3 gene of Hanshenula polymorpha strain and make it a nutritional requirement.
  • Zeo R Zeocin resistance gene
  • the Zeocin resistance gene (Zeo R ) cassette (1192 bp) was obtained by polymerase chain reaction using Zeo resistance ⁇ Forward (SEQ ID NO: 7) and Zeo resistance_Reverse (SEQ ID NO: 8) using the pPICZalphaA vector as a template.
  • the URA3 disruption cassette was prepared using the HpURA3 ⁇ ⁇ N-terminal fragment, Zeocin resistance gene (Zeo R ) cassette, and HpURA3 ⁇ C-terminal fragment prepared above by Fusion PCR. ⁇ 3 crush cassette was used to produce wild type Hanshenula polymorpha DLl), Glucose oxidase (GOD) recombinant strain [(? R /?
  • the present inventors prepared a GAS1 disruption cassette "to generate a disrupted strain lacking the HpGASl gene and introduced it into the mutant strain of Example ⁇ 2-1> in which the U 3 gene was disrupted.
  • ⁇ 45 _ / _ N_Forward (SEQ ID NO: 9) and v3 ⁇ 4o ⁇ 5_N_Reverse_UJF (SEQ ID NO: 9) were used as chromosomal DNA isolated from Hanshenula polymorpha DL1 strain. 10) was used to obtain HpGASl ⁇ ⁇ N-terminal fragment (537 bp), and ⁇ p5SlS ⁇ C- using ⁇ 5 _CJ rward— LUR (SEQ ID NO: 11) and ⁇ A _C— Reverse (SEQ ID NO: 12). Terminal fragments (524 bp) were obtained via polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • pLaclIR3 as a template (Kim et al., J Biol Chem. 281, 6261, 2006) using primer pairs Hp 1 acZ_URA3_N_Forward (SEQ ID NO: 24) and HplacZ_URA3_N_Reverse (SEQ ID NO: 25) for the expression of lacZ-H P URA3-lacZ cassette.
  • N-terminal fragments were obtained by HplacZ_URA3_C_Forward (SEQ ID NO: 26) and Hp lacZ_URA3_C—Reverse (SEQ ID NO: 27) to obtain C-terminal fragments of the lacZ-H P U A3-lacZ cassette by polymerase chain reaction.
  • HpGASlS ⁇ N-terminal fragment, N-terminal fragment of lacZ-HpURA3-lacZ cassette, were obtained by GAS1 disruption cassette _N-terminal fragment by fusion PCR, C-terminal fragment of lacZ-HpURA3-lacZ cassette, HpGASl ⁇ C-terminal Sections were obtained by GAS1 disruption cassette _C ⁇ terminal fragments by fusion PCR (Fig. 5).
  • the prepared GAS1 disrupted cassette ⁇ N-terminal fragment and the GAS1 disrupted cassette _C-terminal fragment were introduced into each of the strains of the URA3 gene fragmented in Example ⁇ 2-1> by homologous recombination, and then, in the SC-URA minimal medium.
  • a mutant strain in which the viable HpGASl gene was disrupted was obtained.
  • the inventors prepared a disruption cassette to introduce a mutant strain in which the UM3 gene of Example ⁇ 2-1> was disrupted to prepare a disrupted strain lacking the H P GAS2 gene.
  • HPGAS2 2 C-terminal fragment (524 bp) was obtained through polymerase chain reaction (PCR) using ⁇ ⁇ 5 ⁇ C_Forward_LU (SEQ ID NO: 15) and ⁇ fi! S2 _Reverse (SEQ ID NO: 16).
  • the N-terminal fragment of the lacZ—HpU A3-lacZ cassette was prepared using the primer pairs HplacZ_URA3_N_Forward (SEQ ID NO: 24) and HplacZ—URA3_N_Reverse (SEQ ID NO: 25).
  • C-terminal fragments of the lacZ-HpURA3-lacZ cassette were obtained by polymerase chain reaction using HplacZ_URA3_C_Forward (SEQ ID NO: 26) and HplacZ_URA3_C_Reverse (SEQ ID NO: 27).
  • HpGAS2 ⁇ ⁇ N ⁇ fragment and N ⁇ terminal fragment of lacZ-HpURA3-lacZ cassette were subjected to fusion PCR.
  • N-terminal fragments were obtained from the shredding cassette
  • H P GAS2 C-terminal fragments were obtained from the GS ⁇ crushing cassette_C-terminal fragments by fusion PCR (FIG. 6).
  • the resulting fragmented cassette ⁇ ⁇ -terminal fragment and the GAS2 fragmented cassette _C-terminal fragment were introduced into each of the strains of the iK4 gene fragmented in Example ⁇ 2-1> through homologous recombination to survive in the SC-URA minimal medium. Mutant strains in which the H P GAS2 gene was disrupted were obtained.
  • the present inventors compared the Hansenula polymorpha wild type DL1 strain with confocal microscopy and optical microscope to confirm the morphological characteristics of the HpGASl gene disruption strain and ⁇ 3 ⁇ 4 ⁇ 5 gene disruption strain prepared in ⁇ Example 2> .
  • the present inventors confirmed the physiological characteristics of the HpGASl gene disruption strain and ⁇ 5 gene disruption strain prepared in ⁇ Example 2> through the sensitivity to the cell wall inhibitor.
  • HpGASl gene disruption or H P GAS2 gene disruption Hansenula polymorpha DL1 wild type strain, HpGASl fragmented strain and HpGAS2 fragmented.
  • the strains were preincubated for 16 hours at 37 ° C at 200 rpm in 3 ml glucose 2% complex media each.
  • the cultured strains were first diluted with 1/10 of 1D in 1 ml of sterile water, and then serialized 1/10 for glucose 2% complex agar medium, 3% Calcofluor White (CFW), a cell wall synthesis inhibitor, 1% Congo Red (C) and osmotic perturbant 0.01% sodium dodecyl sulfate (SDS), respectively, were collected in a glucose 2% complex agar medium containing 1% and incubated at 37 ° C for 3 days.
  • C Calcofluor White
  • SDS osmotic perturbant 0.01% sodium dodecyl sulfate
  • HpGASl gene disruption strains were inhibited in 37% C and 42 0 C glucose 2% agar medium due to the change of cell wall structure, and wild type or even in cell wall synthesis inhibitor-sensitive media and osmotic disturbance-sensitive media. It was confirmed that growth was inhibited and growth rate was slow compared to H P GAS2 crushed strains (FIGS. 1, 2 and 3 of FIG. 9).
  • the inventors of the present invention have found that the cell wall following HpGASl gene disruption and HpGAS2 gene disruption is In order to confirm whether the total protein secretion is increased by the structural change was confirmed by the Bradford assay method.
  • Hanshenula polymorpha DL1 wild type strain,; 3 ⁇ 4 ⁇ A5 gene disruption strain and H P GAS2 gene disruption strain was pre-incubated for 16 hours at 37 ° C at 200 rpm in 3 ml glucose 2> complex medium 50 Incubated in ml glucose 2% complex medium to the initial 0D 600 value 0.3 to the same conditions. Take culture medium at each time of incubation
  • the present inventors confirmed whether the secretion of Glucose oxidase from the GOD producing strain was increased by HpGASl gene disruption.
  • wild type GOD producing strain H. poIymorpha / pIAMOX-GOO
  • HpGASl gene disruption strain polymorpha AffpGASl / pOLV X-GOm
  • HpGAS2 gene disruption strain HpGAS2 gene disruption strain
  • polymorpha o ⁇ 5 / pDLM0X-G0D (H) was incubated in 3 ml glucose 2% complex medium at 200 rpm for 16 hours at 37 ° C, and then the expression of the foreign recombinant protein expressed using the M0X promoter was determined.
  • Hazardous methane was inoculated in 50 ml of 2% complex medium to give an initial 0D 600 value of 0.3, followed by main culture.
  • 0D 600 corresponds to 2
  • the supernatant was secured by centrifugation at 4 ° C for 10 minutes at 13,000 rpm.
  • the supernatant was mixed with 5 ⁇ sample loading buffer, boiled for 5 minutes, and then electrophoresed in two pairs of 83 ⁇ 4> SDS-PAGE gels by dividing the sample in half (ie, the supernatant corresponding to 0D 600 value 1).
  • One of the two gels was stained with Coomassie Brilliant Blue R-250 Staining Solution (BIO-RAD) and bleached with destaining buffer.
  • the other gel was transferred to a PVDF membrane, and then the PVDF membrane was blocked with 5% skim milk solution, and the primary antibody His-probe (Santa Cruz Biotechnology) was diluted 1: 1000 and reacted for 2 hours. After reaction, wash three times for 10 minutes with TBST (50 mM Tris-HCKpH 7.5), 150 mM NaCl and 0.053 ⁇ 4> Tween 20), and then dilute the secondary antibody Anti-Mouse IgG (Sigma) to 1: 3000. The reaction was carried out for 30 minutes. Then, washed with TBST solution three times for 10 minutes and detected by ECL advanceTM Western Blotting Detect ion Kit (AmershamTM-GE Healthcare).
  • HSA secretion of recombinant human serum albumin (HSA) producing strain was increased by HpGASl and HpGAS2 gene disruption. It was. ' Specifically, wild type HSA producing strain (polyniorpha G3T8), HpGASl gene disruption strain (.polyniorpha G3T8 AHpGASl) and HpGAS2 gene disruption strain 07.
  • the supernatant was obtained by centrifugation at 4 ° C. for 10 minutes at 13,000 rpm.
  • the supernatant was mixed with 5 ⁇ sample loading buffer and boiled for less than 5 minutes, and then electrophoresed in two pairs of 1OT SDS—PAGfe gels by dividing the samples in half (ie, the supernatant corresponding to a 0D 600 value of 0.05).
  • One of the two gels was stained with Coomassie Brilliant Blue R_ 2 50 Staining Solution (BIO-RAD) and bleached with destaining buffer.
  • the other gel was transferred to a PVDF membrane, and the PVDF membrane was blocked with a 5% skim milk solution, and the first antibody, Anti-Human Albumin antibody (Sigma), was diluted 1: 10000 and reacted for 2 hours. After reaction, washed three times for 10 minutes with TBST (50 mM Tris-HCKpH 7.5), 150 mM NaCl and 0.05% Tween 20) solution, and then the second antibody, goat ant i -rabbit IgG-HRP (Santa Cruz Biotechnology) 1: Diluted to 5000 and reacted for 1 hour 30 minutes.
  • TBST 50 mM Tris-HCKpH 7.5
  • the present inventors have the same effect as the above experimental example by lacZ-URA3 ⁇ lacZ cassette
  • the lacZ ⁇ URA3-lacZ cassette was removed from the gene disruption strain.
  • chromosomal DNA isolated from Hanshenula polymorpha DL1 strain was used as a template to prepare lacZ-URA3-lacZ removal cassette using ⁇ AWJLForward (SEQ ID NO: 9) and ⁇ A _N—Reverse (SEQ ID NO: 10).
  • H P GAS1 ⁇ ⁇ C-terminal fragments (524 • bp) that could be fused with HpGASl ⁇ N-terminal fragments using A_C—Reverse (SEQ ID NO: 12) were obtained via polymerase chain reaction (PCR).
  • the present inventors produced vectors in which the HpGASl, HpGAS2 and ScGASl genes were cloned, respectively, and introduced them into the HpGASl gene disruption strain (.polyinorpha DL1 AHpGASl ⁇ ?).
  • HpGASl, HpGAS2 gene cassette and Saccharomyces cerevisiae amplified using primers of SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20, respectively, as a template of chromosomal DNA isolated from Hanshenula polymorpha DL1 strain.
  • Chromosomal DNA isolated from BY4741 strain was amplified using primers of SEQ ID NOs: 21 and 22 as templates.
  • the ScGASl gene cassette was cloned into the blunt ends in the pDLGUK vector treated with restriction enzyme _ MssJ (FIG. 1).
  • the pDLGUK vector is a vector with genes ⁇ and ⁇ i? that can be randomly inserted near the telomeres of chromosomal DNA.
  • the transformant is capable of obtaining transformants that grow on SC-URA minimal media because the iffi gene is inserted. Number have.
  • HpGASrA inserted vector pDLGUK-y3 ⁇ 4 ⁇ 7A «
  • HpGAS27 ⁇ inserted vector MM-HpGAS2
  • ⁇ A inserted vector (pDLGUK- ScGASl) and pDLGUK blank were URA3 gene and H P GAS17 ⁇ crushed Hansenula polymorpha DL1 strain (.polymorpha DL1 ⁇ HpGAS ⁇ ⁇ 3) was transformed.
  • the present inventors confirmed the physiological characteristics due to the gene introduced in Experimental Example ⁇ 4-2> by integrating the culture medium diluted in a serial medium on a solid medium.
  • the strain supplemented with the HpGASl gene showed a similar degree of growth as the wild type (4 in FIG. 9), and ⁇ (strain supplemented with the 52 gene was slower than the wild type, but it was found to grow in a sensitive medium (FIG. 9).
  • Saccharomyces cerevisiae Strains supplemented with the GAS1 gene, ScGASl gene did not show growth growth, and showed the same growth as the strain into which the pDLGUK void was introduced (FIGS. 6 and 7 of FIG. 9).
  • scGASl-expressing vector or pDLGUK vaccinated strain was slightly increased than that of untransformed HpGASl crushed strain. It was judged as the same phenomenon reported to grow fast.
  • the present inventors also confirmed growth inhibition by cell wall attenuation by GAS1 gene disruption in Saccharomyces cerevisiae.
  • Saccharomyces cerevisiae strain C. cerevisiae BY4741 Saccharomyces cerevisiae strain C. cerevisiae BY4741
  • ScGASl crushed strain (5. cerevisiae BY4741 ⁇ 5 £) was pre-incubated in 3 ml glucose 2% complex medium at 200 rpm for 16 hours at 30 ° C.
  • the pre-incubated strains were serially maintained at OD 600 value 1 in 1 ml of sterile water. 1/10 dilutions, then 1% glucose 2% agar medium, 0.005% Congo Red (CR) and 0.01% sodium dodecyl sulfate (SDS) ⁇ Were accumulated and incubated for 3 days or 4 days at 30 ° C.
  • the present inventors produced vectors (YEp352ScGAPDHpt -HpGASl, YEp352ScGAPDHpt-HpGAS2), each of which was cloned into the above genes in order to confirm the function of the HpGASl and HpGAS2 genes using Saccharomyces cerevisiae strains. cerevisiae BY4741 AScGASD ⁇ ], respectively.
  • chromosomal DNA isolated from Hanshenula plymorpha DL1 strain Amplified using primers SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, respectively, as a template
  • HpGASl and HpGAS2 gene cassettes were treated with restriction enzyme Xba / and cloned into YEp352ScGAPDHpt vector treated with restriction enzyme XZ / (FIG. 2).
  • the YEp352ScGAPDHpt vector is a vector with a 2 micron origin, a gene and a GAPDH promoter.
  • the transformant can be obtained from the SC-URA medium because the URA3 gene is inserted.
  • the HpGAS] 7 ⁇ inserted vector (YEp352ScGAPDHpt-HpGASl), the HpGAS27 ⁇ inserted vector (YEp352ScGAPDHpt-HpGAS2) and the YEp352ScGAPDHpt blank were scGASl crushed Saccharomyces cerevisiae strain (5. cerevisia SD BY474. Transformants were obtained from SC-URA minimal media, and diluted cell culture fluids were accumulated in sensitive solid media to compare growth.
  • Saccharomyces cerevisiae strain (5. cereP ' s / aeBY4741 ASc 5J / YEp352ScGAPI) Hp1, in which the 5rf 3/4 gene complementing the HpGASl gene was disrupted; -HpGASl) was recovered to a similar extent as wild type (5. cerevisiae BY4741) (Fig. 3, 3), and the strain complementing the HpGAS2 gene (5. cerevisiae BY4741 ASc / Y3 ⁇ 4) 352ScGAPDHpt—HpGAS2) was nocturnal (5. the growth was restored, so similar to the 's / aeBY4741) (4 in Fig.

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Abstract

La présente invention concerne un procédé d'utilisation, dans la production d'une protéine recombinante exogène, d'une variante de souche microbienne ayant une capacité de sécrétion de protéine qui est améliorée en endommageant d'une protéine d'une nouvelle sorte. Plus particulièrement, la séquence de base génique de Hansenula polymorpha a été criblée afin d'identifier un groupe de candidats pour un gène HpGAS codant pour la protéine Gas1 ayant une action bêta-1,3-glucanosyl transglycosylase et, dans ce groupe, un nouveau gène (HpGAS1) a été confirmé, et le gène en question a été délété afin de vérifier la fonction de celui-ci, et par conséquent il a été confirmé qu'il y a des modifications accrues de morphologie cellulaire et une sensibilité accrue à l'inhibiteur de la synthèse de paroi cellulaire, une sécrétion protéique totale accrue et une sécrétion de protéine recombinante accrue de façon marquée, et, par conséquent, l'invention peut être utilisée de façon avantageuse en tant que ressource pour la production par sécrétion, le nettoyage et la collecte d'une protéine recombinante exogène.
PCT/KR2013/003581 2012-06-19 2013-04-25 Souche de levure au gène hpgas1 interrompu et procédé de production d'une protéine recombinante à l'aide de celle-ci WO2013191369A1 (fr)

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KR10-2012-0065686 2012-06-19
KR20120065686 2012-06-19
KR10-2013-0045467 2013-04-24
KR1020130045467A KR101498012B1 (ko) 2012-06-19 2013-04-24 HpGAS1 유전자 파쇄 효모 균주 및 이를 이용한 재조합 단백질의 생산 방법

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WO1998001473A1 (fr) * 1996-07-05 1998-01-15 Novo Nordisk A/S Procede de production de precurseurs de l'insuline, precurseurs d'analogues de l'insuline et peptides insulinoides
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