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WO2024144431A1 - Nouvelle souche productrice de escherichia coli produisant une protéine hybride cbd-hs-es-glp1, et procédé de production de polypeptide glp-1 - Google Patents

Nouvelle souche productrice de escherichia coli produisant une protéine hybride cbd-hs-es-glp1, et procédé de production de polypeptide glp-1 Download PDF

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Publication number
WO2024144431A1
WO2024144431A1 PCT/RU2023/050300 RU2023050300W WO2024144431A1 WO 2024144431 A1 WO2024144431 A1 WO 2024144431A1 RU 2023050300 W RU2023050300 W RU 2023050300W WO 2024144431 A1 WO2024144431 A1 WO 2024144431A1
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cbd
glp1
purification
polypeptide
plyss
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Russian (ru)
Inventor
Алексей Алексеевич ЗИНЧЕНКО
Дмитрий Александрович МАКАРОВ
Игорь Валентинович МЯГКИХ
Даниил Михайлович ПАВЛЕНКО
Василий Николаевич СТЕПАНЕНКО
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Avva Pharmaceuticals Ltd
Federal State Budgetary Institution Of Science MM Shemyakin YuA Ovchinnikov Institute Of Bioorganic Chemistry Of Russian Academy Of Sciences Ibch Ras
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Avva Pharmaceuticals Ltd
Federal State Budgetary Institution Of Science MM Shemyakin YuA Ovchinnikov Institute Of Bioorganic Chemistry Of Russian Academy Of Sciences Ibch Ras
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    • 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
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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

Definitions

  • the present invention relates to the field of genetic and protein engineering and can be used in medicine and the pharmaceutical industry. More specifically, the present invention relates to a new producer strain BL21(DE3) pLysS/CBD-HS-ES-GLP1, producing a CBD-HS-ES-GLP1 fusion protein, as well as a method for producing GLP-1 polypeptide using the said strain.
  • the GLP-1 polypeptide having the sequence SEQ ID NO 1 is one of the incretins that lowers glucose levels by modulating insulin secretion by liver beta cells [Baggio LL, Drucker DJ (2007) Biology of incretins: GLP-1 and GIP. Gastroenterology 132:2131–2157].
  • GLP-1 and its analogues are used to stimulate insulin secretion in patients with non-insulin-dependent diabetes. Moreover, GLP-1 and its analogues inhibit glucagon secretion, which leads to a significant decrease in blood glucose levels.
  • a known method for producing the GLP-1 polypeptide in a construct containing several copies of the corresponding gene [CA3057252, C07K14/605, publ. 09/27/2018].
  • the disadvantage of this method is the use of two proteases at once at the stage of cleavage of the fusion protein, which increases the cost of the production process and complicates further purification.
  • this method does not propose an approach to cleaning, which, due to the features of the design used, will be complex and resource-intensive.
  • the present inventors have previously developed a unique CBD-HS-ES-GLP1 fusion protein with the amino acid sequence SEQ ID NO 2, intended to produce a GLP-1 polypeptide, overcoming the disadvantages of prior art methods for producing a GLP-1 polypeptide. Also, the authors of the present invention have previously developed a recombinant plasmid pET23b-CBD-HS-ES-GLP1 for the expression of the specified hybrid protein.
  • the unique hybrid protein CBD-HS-ES-GLP1 previously developed by the authors of the present invention, designed to produce the GLP-1 polypeptide, and the recombinant plasmid pET23b-CBD-HS-ES-GLP1 provide the production of the GLP-1 polypeptide with high yield and high purity.
  • This technical problem is solved by the present invention by providing a new highly productive bacterial strain-producer BL21(DE3) pLysS/CBD-HS-ES-GLPl, producing a hybrid protein CBD-HS-ES-GLP1, as well as a method for producing the GLP-1 polypeptide, allowing obtain GLP-1 polypeptide with increased yield and purity.
  • a known strain of E. coli cells is BL21(DE3)/pLysS, containing the plasmid pLysS which has the arrangement of elements as shown in Fig. 9, which is used for highly efficient gene expression under the control of the T7 promoter and allows achieving a high level of target gene expression when inducing with IPTG (isopropyl-PDl-thiogalactoprianoside) or lactose.
  • Cells of this strain have the ability, in the presence of an inducer (IPTG or lactose), to predominantly express recombinant proteins and a minimal amount of their own proteins.
  • This strain was used in the present invention as a parent strain for transfection with the target plasmid pET23b-CBD-HS-ES-GLPl and obtaining a new highly productive bacterial a new producer strain BL21(DE3) pLysS/CBD-HS-ES-GLPl, producing a hybrid protein CBD-HS-ES-GLPE
  • the unexpected technical result of the present invention discovered by the authors is to achieve an increased yield of a hybrid protein containing the GLP-1 polypeptide, constituting at least 40% of the total cell protein, reducing background expression from the pET23b-CBD-HS-ES-plasmid promoter GLP1, which reduces the parasitic production of the hybrid protein at the stage of inoculation and fermentation by 80% and increases its stability to elimination in culture by 50%.
  • the present invention provides the production of GLP-1 polypeptide with a purity of 99%.
  • the present invention relates to a new producer strain BL21(DE3) pLysS/CBD-HS-ES-GLP1 to produce a GLP1 polypeptide with the amino acid sequence SEQ ID NO 1, producing a hybrid protein CBD-HS-ES-GLP1 with the amino acid sequence SEQ ⁇ NO 2 wherein said Escherichia coli strain BL21(DE3) pLysS/CBD-HS-ES-GLP1 contains recombinant plasmid DNA pLysS, which has the sequence SEQ ID NO 284 and the arrangement of elements shown in FIG. 9, and plasmid DNA pET23b-CBD-HS-ES-GLP1 for expression of the CBD-HS-ES-GLP1 fusion protein, consisting of the following key genetic elements:
  • fl ori bacteriophage fl
  • the specified hybrid protein CBD-HS-ES-GLP1 with the amino acid sequence SEQ ID NO 2 contains the amino acid sequence X1X2YX3, a chitin-binding domain (CBD), a specific recognition site for enteropeptidase (ES) , a hexahistidine domain (HS), GLP-1 polypeptide having the amino acid sequence of SEQ ID NO 1, where X1X2YX3 is an amino acid sequence in which Xi is selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V, Xr is selected from K or N, and X3 is absent or selected from H or Q.
  • the CBD-HS-ES-GLP1 fusion protein has the amino acid sequence X1X2YX3, which is VKY.
  • the CBD-HS-ES-GLP1 fusion protein has the amino acid sequence X1X2YX3, which is VNY.
  • said recombinant plasmid DNA pET23b-CBD-HS-ES-GLPl is a recombinant plasmid DNA pET-parB-CBD-HS-ES-GLPl, has a length of 4391 and. o., contains the hok/sok module, which stabilizes plasmids [T, Gerdes K. Mechanism of post-segregational killing by the hok/sok system of plasmid R1. Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene. J Mol Biol. 1992 Jan 5;223(l):41-54. doi: 10.1016/0022-2836(92)90714-u.
  • the Escherichia coli strain according to this embodiment is also referred to as Escherichia coli BL21(DE3) pLysS/KanR-ParB/CBD-HS-ES-GLP 1
  • said recombinant plasmid DNA pET23b-CBD-HS-ES-GLPl is a recombinant plasmid DNA pET23bKanR-CBD-HS-ES-GLPl, has a length of 3820 and. Thus, it contains the kanamycin antibiotic resistance gene KanR as an antibiotic resistance gene, and the location of its genetic elements is shown in Fig. 2.
  • the Escherichia coli strain according to this embodiment is also referred to as BL21(DE3)/pLysS/KanR/CBD-HS-ES-GLPl.
  • the present invention also relates to a method for producing a GLP1 polypeptide having the amino acid sequence SEQ ID NO 1, comprising the steps of: a) cultivating the producer strain Escherichia coli BL21(DE3) pLysS/CBD- HS-ES-GLP1 according to the present invention with the production of cell biomass of a producer strain producing a hybrid protein CBD-HS-ES-GLP1 with the amino acid sequence SEQ ID NO 2; b) isolating said CBD-HS-ES-GLP1 hybrid protein from the cell biomass of the producer strain obtained in step a); c) enzymatic cleavage of said CBD-HS-ES-GLP1 fusion protein obtained in step b) to form a GLP-1 polypeptide with the amino acid sequence SEQ ID NO 1; d) purifying the GLP-1 polypeptide obtained in step c).
  • step b) involves separating cells from the culture fluid, disintegrating the cells, isolating inclusion bodies from the resulting disintegrate, and solubilizing the inclusion bodies.
  • ammonium sulfate is added in step b) when separating the inclusion bodies from the resulting disintegrate.
  • the cells are separated from the culture liquid, the cells are disintegrated, and the hybrid protein is isolated from the soluble fraction of the cell disintegrate.
  • the cells are separated from the culture liquid, the cells are disintegrated, and the hybrid protein is isolated from the soluble fraction of the cell disintegrate.
  • step b) purifies the CBD-HS-ES-GLP1 fusion protein by chromatography on a metal chelate sorbent.
  • step b) the purification of the CBD-HS-ES-GLP1 fusion protein is carried out by chromatography on a cation exchange sorbent.
  • step b) purifies the CBD-HS-ES-GLP1 fusion protein by chromatography using anion exchange chromatography.
  • step b) purifies the CBD-HS-ES-GLP1 fusion protein by chromatography using hydrophobic interaction chromatography.
  • step b) purifies the CBD-HS-ES-GLP1 fusion protein by chromatography using affinity chromatography.
  • step c) the cleavage of the fusion protein is carried out by enteropeptidase.
  • step d) purifies the GLP-1 polypeptide using a single chromatography step.
  • step d) purification of the GLP-1 polypeptide is carried out using several chromatographic steps.
  • step d) purifies the GLP-1 polypeptide using reverse phase chromatography.
  • step d) purifies the GLP-1 polypeptide using gel filtration chromatography.
  • step d) purifies the GLP-1 polypeptide by ammonium sulfate precipitation and buffer extraction of impurities.
  • step d) purifies the GLP-1 polypeptide by chromatography on a chitin-based sorbent.
  • step d) purifies the GLP-1 polypeptide by chromatography using cation exchange chromatography.
  • step d) purifies the GLP- polypeptide 1 is carried out chromatographically using hydrophobic chromatography.
  • step d) purifies the GLP-1 polypeptide by chromatography using metal chelate chromatography.
  • step d) purifies the GLP-1 polypeptide by chromatography using affinity chromatography.
  • Fig. 1 Map of the plasmid pET-parB-CBD-HS-ES-GLPl, where the given designations have the following meanings: fl ori - origin of replication of the bacteriophage fl, KanR - kanamycin resistance gene (aminoglycoside phosphotransferase gene), ori - origin of replication colEl, T7 promoter - promoter of bacteriophage T7, ragB - hok/sok locus, CBD-HS-ES-GLP1 - hybrid protein gene CBD-HS-ES-GLP1, including the GLP-1 polypeptide, hexahistidine and chitin-binding sequences, T7 terminator - terminator bacteriophage T7.
  • fl ori - origin of replication of the bacteriophage fl KanR - kanamycin resistance gene (aminoglycoside phosphotransferase gene), ori - origin of replication colEl, T7 promoter
  • Fig. 3 Electrophoretic analysis of total cell lysate, where the given designations have the following meanings: 1 - at the time of induction, 2 - 2 hours after induction, 3 - 4 hours after induction, 4 - 5 hours after induction, 5 - molecular weight standards, Pierce Unstained Protein MW Marker, Thermo, cat # 26610.
  • Fig. 4 Electrophoretic analysis of total cell lysate, where the given designations have the following meanings: 1 - at the time of induction, 2 - 2 hours after induction, 3 - 4 hours after induction, 4 - 5 hours after induction, 5 - molecular weight standards, Pierce Unstained Protein MW Marker, Thermo, cat # 26610.
  • Fig. 5 Electrophoretic analysis of the hybrid protein CBD-HS-ES-GLP1, where the given designations have the following meanings: 1 - molecular weight standards, Pierce Unstained Protein MW Marker, Thermo, cat # 26610, 2 - 3 - hybrid protein CBD-HS-ES -GLP1 before incubation with enteropeptidase, 4 - 5 - reaction mixture containing the GLP-1 polypeptide obtained after incubation with enteropeptidase.
  • Fig. 9 Map of the pLysS plasmid, where the given designations have the following meanings: cat promoter - promoter of the E. coli cat gene, p15A ori - medium-copy origin of replication, allowing the use of a second plasmid containing ColEl or MB1 origin (pET vectors) in E. coli cells , T7 lysosyme - lysozyme from bacteriophage T7, inhibitor of T7 RNA polymerase, T7 F3.8 promoter - weak promoter for T7 RNA polymerase, CmR - gene encoding chloramphenicol acetyltransferase (cat), which determines resistance to chloramphenicol.
  • Producer strain of Escherichia coli BL21(DE3) pLysS/CBD-HS-ES-GLPl producing the hybrid protein CBD-HS-ES-GLP1 (SEQ ID NO 2), in addition to the specified recombinant DNA plasmid pET23b-CBD-HS-ES-GLPl contains plasmid pLysS, which has the sequence of SEQ ID NO 284 and the arrangement of elements shown in FIG. 9.
  • Cultivation of Escherichia coli BL21(DE3) pLysS/CBD-HS-ES-GLPl cells in growth medium is carried out for at least 6 hours.
  • Induction of recombinant protein biosynthesis by BL21(DE3) pLysS/CBD-HS-ES-GLP1 cells is carried out 2-5 hours after the start of cultivation using isopropyl-P-D-1-thio galactoprianoside or lactose.
  • the separation of the culture liquid from the cells is carried out by centrifugation.
  • Cell disintegration is carried out using an ultrasonic disintegrator.
  • Isolation of inclusion bodies from the disintegrate is carried out by centrifugation.
  • Isolation of the CBD-HS-ES-GLP1 hybrid protein from the resulting solubilized inclusion bodies is carried out chromatographically on a metal-chelate, cation-exchange, anion-exchange, hydrophobic, affinity or chitin-based sorbent, or by precipitation with ammonium sulfate or by lowering the pH below the isoelectric point and extraction of impurities with a buffer solution.
  • GLP-1 polypeptide from the CBD-HS-ES-GLP1 hybrid protein is carried out by enzymatic hydrolysis with enteropeptidase.
  • enteropeptidase in this document means a proteolytic enzyme with code EC 3.4.21.9, as well as all its fragments and analogs that have specific proteolytic activity against fragments of amino acid sequences including -Asp-Asp-Asp-Asp-Lys- (SEQ ID NO 6) and -Asp-Asp-Asp- Asp-Arg- (SEQ ID NO 4).
  • the posed technical problem of this invention can be solved by constructing the expression plasmid pET-parB-CBD-HS-ES-GLPl with a length of 4391 and. o., ensuring the expression of the hybrid protein CBD-HS-ES-GLP1 in Escherichia Coli cells transformed with the specified plasmid.
  • the posed technical problem of this invention can also be solved by constructing the expression plasmid pET23bKanR-CBD-HS-ES-GLPl with a length of 3820 and. o., ensuring the expression of the hybrid protein CBD-HS-ES-GLP1 in Escherichia Coli cells transformed with the specified plasmid.
  • KanR antibiotic kanamycin
  • the starting material for creating the producer strain according to the invention is the E. coli strain BL21(DE3)/pLysS, known from the prior art.
  • Physico-biological characteristics cells grow at temperatures from 4°C to 40°C with an optimal pH value of 6.8 to 7.5. Used as a nitrogen source mineral salts in ammonium form, and organic compounds, including peptone, tryptone, yeast extract, amino acids, etc. Amino acids, glycerin, and carbohydrates are used as a carbon source.
  • the starting material for creating the producer strain of the invention is the Coli strain BL21 (DE3)/pLysS, known from the prior art.
  • an electroporation method known in the art and incorporated herein by reference is used to introduce said plasmid into cells of the Coli strain BL21 (DE3)/pLysS.
  • transformation methods known in the art for example, the polyethylene glycol method, the calcium chloride method, can be used to introduce the plasmid into E. Coli BL21 (DE3)/pLysS cells.
  • the plasmid shown in FIG. 2 for introducing into E. Coli BL21 (DE3)/pLysS cells the plasmid shown in FIG. 2, other transformation methods may be used that are not explicitly mentioned in this description, which Some are currently known in the art or will be created later.
  • one skilled in the art based on the existing level of knowledge, can select the most optimal method for transforming cells.
  • Transformed cells are seeded onto Petri dishes with an agar medium with the addition of the selection agent kanamycin sulfate to a final concentration of kanamycin sulfate of 50 ⁇ g/ml cell. From the kanamycin-resistant clones, the DNA of the plasmid pET23bKanR-CBD-HS-ES-GLPl is isolated, which is analyzed by sequencing.
  • Morphological characteristics rod-shaped cells, gram-negative, non-spore-bearing.
  • Physico-biological characteristics cells grow at temperatures from 4°C to 40°C with an optimal pH value of 6.8 to 7.5. Both mineral salts in ammonium form and organic compounds, including peptone, tryptone, yeast extract, amino acids, etc. are used as a source of nitrogen. Amino acids, glycerin, and carbohydrates are used as a carbon source.
  • the method for producing the CBD-HS-ES-GLP1 hybrid protein involves cultivating cells of the producer strain E. coli BL21(DE3) pLysS/KanR-ParB-CBD-HS-ES-GLPl, obtained by transforming E. coli BL21(DE3) pLysS cells disclosed in FIG. 1 plasmid length 4391 and. o., consisting of the specified key genetic elements, on a growth medium to obtain a culture liquid, separation of cell biomass from the culture liquid, cell disintegration and isolation of the CBD-HS-ES-GLP1 hybrid protein from the cell disintegrate.
  • Isolation from the disintegrate can be carried out by isolating the inclusion bodies from the resulting disintegrate, solubilizing the inclusion bodies and isolating the CBD-HS-ES-GLP1 hybrid protein from them, or by isolating the CBD-HS-ES-GLP1 hybrid protein from the liquid fraction of the cellular disintegrate.
  • One skilled in the art will understand that it is possible to achieve higher production of the CBD-HS-ES-GLP1 hybrid protein when using fermenters. It is also clear to the specialist that a further increase in production is possible by improving mass transfer and oxygen supply to the fermenter.
  • the induction of recombinant protein biosynthesis in E. coli BL21(DE3) pLysS/KanR-ParB-CBD-HS-ES-GLPl cells is carried out at 3 hours of culture using isopropyl-P-D-l-thiogalactoprianoside.
  • the induction of recombinant protein biosynthesis in E. coli BL21(DE3) pLysS/KanR-ParB-CBD-HS-ES-GLP1 cells is carried out at 5 hours of culture using lactose.
  • the separation of the culture fluid from the cells is accomplished by centrifugation.
  • cell disintegration is carried out using an ultrasonic disintegrator.
  • the separation of inclusion bodies from the disintegrate is carried out by centrifugation.
  • the isolation of the CBD-HS-ES-GLP1 fusion protein from the resulting solubilized inclusion bodies is carried out chromatographically. In more preferred embodiments, but not limited to them, the isolation of the CBD-HS-ES-GLP1 fusion protein from the solubilized inclusion bodies is carried out on a metal chelate chromatographic sorbent.
  • Cleavage of the CBD-HS-ES-GLP1 hybrid protein in a preferred embodiment, but also without limitation, is carried out using enteropeptidase, based on a ratio of 1 unit. enzyme per 1 mg of hybrid protein.
  • further purification of the GLP-1 polypeptide is carried out using chromatographic methods.
  • purification of the GLP-1 polypeptide is carried out using precipitation at a pH value of 5.0-6.0 and extraction of impurities with a buffer solution.
  • oligonucleotides Chemical synthesis of oligonucleotides is performed by the solid-phase phosphoamidite method on an ASM-102U DNA synthesizer (BIOSSET, Novosibirsk) with the extension of the oligonucleotide chain in the direction from the 3'-end to the 5'-end using protected phosphomidites - 5'-dimethoxytrityl-M -acyl-2'-deoxynucleoside-3'-O-(P-cyanoethyl-diisopropylamino)-phosphites activated by tetrazole.
  • BIOSSET Novosibirsk
  • the synthesis is carried out on a scale of 0.5-0.7 ⁇ mol, using porous glass (pore size 500 A) as a carrier, to which the first nucleoside unit is attached via a 3'-succinate bond (load 20-30 ⁇ mol/g).
  • the synthetic cycle of the standard phosphoamidite method is used.
  • plasmid DNA pET23b (450 ⁇ g, 150 pmol) is treated in 6 ml of buffer 20 mM Tris acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 100 ⁇ g/ml BSA with restriction enzyme Xhol (1000 act. units), and then - in 6 ml of buffer 100 mM NaCl, 50 mM Tris-HC1, 10 mM MgCb, 100 ⁇ g/ml BSA with Ndel restriction enzyme (1000 act. units) for 1 hour at 37°C.
  • the vector fragment after electrophoresis in a 15% agarose gel is cut out from the gel and transferred to 30 ml of NT buffer, dissolved at 50 °C for 5-10 min and applied to a NucleoSpinExtractll column. Wash with NT 3 buffer and elute with 7.5 ⁇ l NE buffer.
  • the following variants of the hybrid protein were obtained, having the amino acid sequences SEQ ID NO 146 - SEQ ID NO 283
  • the resulting variants of synthetic sequences SEQ ID NO 8 - SEQ ID NO 145 are individually treated in 40 ⁇ l of buffer 20 mM Tris acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 100 ⁇ g/ml BSA with restriction enzyme Xhol (10 units of act.) , and then in 40 ⁇ l of buffer 100 mM NaCl, 50 mM Tris-HC1, 10 mM MgCb, 100 ⁇ g/ml BSA with Ndel restriction enzyme (10 units of activity) for 1 hour at 37°C.
  • the synthetic fragment is cut out from the gel and transferred into 200 ⁇ l of NT buffer, dissolved at 50°C for 5-10 minutes and applied to a NucleoSpinExtractll column. Wash with NT 3 buffer and elute with 50 ⁇ l NE buffer.
  • the DNA variants of the plasmid pET23b-CBD-HS-ES-GLPl obtained in example 1 in an amount of 3 ⁇ g (1 pmol) are each individually treated in 40 ⁇ l of buffer 20 mM Tris acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 100 ⁇ g /ml BSA restriction enzyme Psil (10 active units), and then in 40 ⁇ l of buffer 100 mM NaCl, 50 mM Tris-HC1, 10 mM MgCb, 100 ⁇ g/ml BSA with restriction enzyme Pcil (10 active units) for 1 hour at 37 °C.
  • the vector fragment is excised from the gel and transferred to 200 ⁇ l of NT buffer, dissolved at 50 °C for 5-10 min and applied to a NucleoSpinExtractll column. Wash with NT 3 buffer and elute with 50 ⁇ l NE buffer.
  • the vector fragment after electrophoresis in a 15% agarose gel is cut out from the gel and transferred to 3 ml of NT buffer, dissolved at 50 °C for 5-10 min and applied to a NucleoSpinExtractll column. Wash with NT 3 buffer and elute with 7.5 ml NE buffer.
  • the resulting fragment of plasmid pET28a described above in an amount of 2 pmol is added separately to solutions of 1 ⁇ g obtained from the DNA of plasmid pET23b-CBD-HS-ES-GLP1, the vector fragments described above in 10 ⁇ l of buffer (20 mM Tris-HC1, pH 7, 56, 10 mM MgC12, 0.2 mM gATP, 10 mM dithiothreitol) and ligated with 10 units. Act. T4-DNA ligase for 12 hours at 10°C.
  • plasmid DNA is used to transform competent E. coli BL21 (DE3) cells. Transformants are plated on plates with LB agar medium, to which kanamycin sulfate is added to a final kanamycin sulfate concentration of 50 ⁇ g/ml.
  • the DNA of the plasmid pET23bKanR-CBD-HS-ES-GLP 1 is isolated from the clones. Recombinants are screened using sequencing.
  • Example 3 Construction of an expression plasmid with an enhancement module stability of the genetic design
  • the DNA variants of the plasmid pET23bKanR-CBD-HS-ES-GLPl obtained in example 2 in an amount of 3 ⁇ g (1 pmol) are each individually treated in 40 ⁇ l of buffer 20 mM Tris acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 100 ⁇ g /ml BSA restriction enzyme Hsha! (10 active units), and then in 40 ⁇ l of buffer 100 mM NaCl, 50 mM Tris-HC1, 10 mM MgCb, 100 ⁇ g/ml BSA with restriction enzyme Pcil (10 active units) for 1 hour at 37° WITH.
  • the vector fragment is excised from the gel and transferred to 200 ⁇ l of NT buffer, dissolved at 50 °C for 5-10 min and applied to a NucleoSpinExtractll column. Wash with NT 3 buffer and elute with 50 ⁇ l NE buffer.
  • the synthetic sequence SEQ ID NO 7 (60 pmol) is treated in 6 ml of buffer 20 mM Tris acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 100 ⁇ g/ml BSA with the restriction enzyme Hsha! (1000 active units), and then in 6 ml of buffer 100 mM NaCl, 50 mM Tris-HC1, 10 mM MgCb, 100 ⁇ g/ml BSA with restriction enzyme Pcil (1000 active units) for 1 hour at 37° WITH.
  • the vector fragment after electrophoresis in a 15% agarose gel is cut out from the gel and transferred to 3 ml of NT buffer, dissolved at 50 °C for 5-10 min and applied to a NucleoSpinExtractll column. Wash with NT 3 buffer and elute with 50 ⁇ l NE buffer.
  • the above-described fragment of the synthetic sequence SEQ ID NO 145 in an amount of 2 pmol is added separately to solutions of 1 ⁇ g obtained from the DNA of the plasmid pET23b-KanR-CBD-HS-ES-GLPl, the vector fragments described above in 10 ⁇ l of buffer (20 mM Tris -HC1, pH 7.56, 10 mM MgC12, 0.2 mM gATP, 10 mM dithiothreitol) and ligated with 10 units. Act. T4-DNA ligase for 12 hours at 10°C.
  • Plasmid DNA pET-parB-CBD-HS-ES-GLP 1 is isolated from the clones. Recombinants are screened using sequencing.
  • Example 4 Obtaining the producer strain E. coli BL21(DE3) pLysS/KanR-ParB-CBD-HS-ES-GLP1 and characterizing its productivity
  • the producer strain E. coli BL21(DE3) pLysS/KanR-ParB-CBD-HS-ES-GLPl is obtained by transforming competent cells of E. coli BL21(DE3)/pLysS with the plasmid pET-parB-CBD-HS-ES-GLP1, the production of which described in example 3.
  • the producer strain E. coli BL21(DE3) pLysS/KanR-ParB-CBD-HS-ES-GLPl is cultivated at 37°C in 100 ml of liquid nutrient medium LB with the addition of kanamycin sulfate to a final concentration of 50 ⁇ g/ml kanamycin sulfate in for 3 hours in Erlenmeyer flasks (1 L, Corning) in an orbital shaker-incubator with a rotation speed of 220 rpm until the optical density of the culture liquid at a wavelength of 600 nm is 0.7-0.8 units.
  • the biosynthesis of the recombinant protein is induced by adding isopropyl-P-D-l-thio galactoprianoside to a final concentration of isopropyl-P-D- 1 -thio galactoprianoside 0.5 mM and incubate for 4 hours. Every hour, a sample of 2 ml is taken, the amount of culture , corresponding to 1 ml, is centrifuged for 10 minutes at 6000 rpm.
  • the precipitated cells are transferred to 100 ⁇ l of lysis buffer with bromophenol blue dye, with the addition of 2-mercaptoethanol, incubated for 5 minutes at 98 °C, 3 ⁇ l aliquots are used for electrophoresis in 14% SDS-PAGE.
  • the gel is stained by adding 0.1% Coomassie R-250 solution and scanned using a Shimadzu CS-930 densitometer. The results are presented in Fig. 3. 4 - cell lysate, 5 - molecular weight standards.
  • the yield of the hybrid protein containing the GLP-1 polypeptide, according to the results of densitometric analysis, is 40% relative to the total cell protein.
  • the producer strain E. coli BL21 (DE3)/KanR/CBD-HS-ES-GLPl is cultivated at 37°C in 100 ml of liquid nutrient medium LB with the addition of kanamycin sulfate to a final concentration of 50 ⁇ g/ml kanamycin sulfate for 3 hours in Erlenmeyer flasks (1 L, Corning) in an orbital shaker-incubator with a rotation speed of 220 rpm until the optical density of the culture liquid is reached at a wavelength of 600 nm 0.7-0.8 units.

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Abstract

La présente invention se rapporte au domaine du génie génétique et protéinique, et peut être utilisée en médecine et dans l'industrie pharmaceutique. Plus précisément, la présente invention concerne une nouvelle souche productrice de BL21(DE3) pLysS/CBD-HS-ES-GLP1 produisant une protéine hybride CBD-HS-ES-GLP1, ainsi qu'un procédé de production de polypeptide GLP-1 à l'aide de ladite souche.
PCT/RU2023/050300 2022-12-29 2023-12-19 Nouvelle souche productrice de escherichia coli produisant une protéine hybride cbd-hs-es-glp1, et procédé de production de polypeptide glp-1 Ceased WO2024144431A1 (fr)

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EA202293542 EA050533B1 (ru) 2022-12-29 Новый штамм-продуцент escherichia coli, продуцирующий гибридный белок cbd-hs-es-glp1, и способ получения полипептида glp-1

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EA013796B1 (ru) * 2005-09-22 2010-06-30 Байокомпатиблз Юк Лтд. Слитые полипептиды, содержащие glp-1 (глюкагон-подобный пептид-1), с повышенной устойчивостью к пептидазам
WO2018136572A1 (fr) * 2017-01-18 2018-07-26 Savior Lifetec Corporation Construction d'expression et procédé de production de protéines d'intérêt
US20200024321A1 (en) * 2017-03-20 2020-01-23 Lupin Limited Expression and large-scale production of peptides
US20220089669A1 (en) * 2018-10-22 2022-03-24 Janssen Pharmaceutica Nv Glucagon like peptide 1 (glp1)-growth differentiation factor 15 (gdf15) fusion proteins and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EA013796B1 (ru) * 2005-09-22 2010-06-30 Байокомпатиблз Юк Лтд. Слитые полипептиды, содержащие glp-1 (глюкагон-подобный пептид-1), с повышенной устойчивостью к пептидазам
WO2018136572A1 (fr) * 2017-01-18 2018-07-26 Savior Lifetec Corporation Construction d'expression et procédé de production de protéines d'intérêt
US20200024321A1 (en) * 2017-03-20 2020-01-23 Lupin Limited Expression and large-scale production of peptides
US20220089669A1 (en) * 2018-10-22 2022-03-24 Janssen Pharmaceutica Nv Glucagon like peptide 1 (glp1)-growth differentiation factor 15 (gdf15) fusion proteins and uses thereof

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