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WO2024210747A1 - Polypeptides de remplacement de la gélatine - Google Patents

Polypeptides de remplacement de la gélatine Download PDF

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Publication number
WO2024210747A1
WO2024210747A1 PCT/NL2024/050170 NL2024050170W WO2024210747A1 WO 2024210747 A1 WO2024210747 A1 WO 2024210747A1 NL 2024050170 W NL2024050170 W NL 2024050170W WO 2024210747 A1 WO2024210747 A1 WO 2024210747A1
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WIPO (PCT)
Prior art keywords
polypeptide
block
culture medium
host cells
optionally
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Pending
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PCT/NL2024/050170
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English (en)
Inventor
Marc Willem Theodoor Werten
Zheng YOU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wageningen Universiteit
Tate and Lyle Solutions USA LLC
Original Assignee
Wageningen Universiteit
Tate and Lyle Solutions USA LLC
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Priority claimed from EP23167140.5A external-priority patent/EP4442697A1/fr
Application filed by Wageningen Universiteit, Tate and Lyle Solutions USA LLC filed Critical Wageningen Universiteit
Publication of WO2024210747A1 publication Critical patent/WO2024210747A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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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
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • 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
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the invention is in the field of gelatin-replacement polypeptides.
  • the invention pertains to tri- and multi-block polypeptides capable of being produced in yeast host cells, and capable of gelling.
  • Natural gelatin is regularly obtained by heat denaturation, i.e., a disintegration of the triple helix structure, after hydrolysis of the intermolecular covalent crosslinks in fibrillar collagen from animal sources, notably type I and type III collagen from bovine or porcine origin.
  • Collagen is made up of three polypeptide strands. Each of these is a left-handed extended helix, and together they are twisted into a triple helix.
  • Animal collagen is held together by intermolecular crosslinks that are formed after formation of the triple helical collagen molecules themselves and their organization into fibrils, the degree of crosslinking increasing with the age of the tissue.
  • crosslinks must be largely hydrolyzed in order to make the collagen and - after heat denaturation - the resulting gelatin, soluble.
  • the necessary hydrolysis brought about at extreme pH, also results in unwanted and largely uncontrollable partial hydrolysis of the collagen backbone, and (when treated at high pH) in deamidation and shifting of the isoelectric point.
  • the animal origin is seen as a risk, in view of the chances that the animal collagen may carry transmissible disease agents, e.g., spongiform encephalopathy prions, such as the well-known bovine spongiform encephalopathy (BSE), which is seriously suspected to be linked to the fatal neurological disease of Creutzfeldt-Jakob in humans.
  • BSE bovine spongiform encephalopathy
  • this is a critical issue, which has resulted in regulatory restrictions on the origin of the gelatins used in these products.
  • a tendency in society is to try and reduce cattle breeding in view of environmental concerns.
  • an emerging desire is to replace proteins traditionally derived from animal sources, by proteins produced in an animal-free manner, yet with the same functionalities.
  • Gelatin is an outstanding example of proteins for which it is sought to meet this desire.
  • the foregoing comes in addition to previously existing reasons to avoid animal-produced proteins, such as reasons based on animal welfare (vegetarian or vegan lifestyle) or religious reasons.
  • yeast -derived, fungal or bacterial products can be considered to be suitable replacements as being, inter alia, vegan as well as kosher or halal.
  • WO 2009/151327 proposes a block co-polypeptide comprising at least two trimerizing blocks and at least one spacer block.
  • the disclosure emphasizes the tunability of the polypeptide structures to be produced, and represents that the block co-polypeptide is capable of forming a gel, and is useful as an animal-free, biocompatible substitute for gelatin.
  • Background art related to these triblock designed polypeptides is Werten et al., Biomacromolecules (2009), Vol.10, pp. 1106-1113.
  • Producing artificial, mammahan e.g., bovine or porcine
  • polypeptide also comes with a disadvantage.
  • it is desired to avoid animal gelatin.
  • the public at large prefers natural products over artificial products, particularly with respect to food.
  • Pichia pastoris provides a desirable platform for the production of recombinant gelatins. Nonetheless, it is quite challenging to find natural gelatin-like sequences that actually can be produced as intact proteins in Pichia pastoris. This relates to the phenomenon that many polypeptide sequences will be susceptible to proteolytic cleavage by protein processing enzymes naturally present in Pichia pastoris.
  • a gel-forming polypeptide suitable as a gelatin replacement that can be produced in an animal-free manner, preferably in Pichia pastoris, yet comprising, preferably consisting of natural peptide sequences.
  • the invention presents, in one aspect, a polypeptide comprising at least one oligopeptide selected from the group consisting of the oligopeptides of formula (II), (HI), (IV), and (V):
  • GPKGEPGSPGEN II
  • GPKGNSGEP IH
  • GSPGEQ preferably a polypeptide having an amino acid sequence comprising at least one of each of said oligopeptides of formula (II), (III), (IV), and (V), more preferably a polypeptide having the sequence of formula (I) (GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ)q (I) wherein q is an integer of from 1 to 40.
  • the invention resides in a block co-polypeptide having a structure (Tbiock-Gbiock)n-Tbiock, wherein: n is an integer of from 1 to 18;
  • Gbiock represents a peptide block defined in accordance with the polypeptide defined hereinabove, the total number of amino acid residues in (Tbiock-Gbiock)n-Tbiock preferably being at most 1500, more preferably at most 1200, and Gbiock more preferably satisfying the structure (GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ)q, wherein q is an integer of from 1 to 40; the capital letter designations defining the peptide blocks in accordance with the conventional one-letter nomenclature for amino acids.
  • the invention provides a hydrogel comprising a block co-polypeptide as defined in the preceding paragraph.
  • the invention resides in a nucleic acid molecule encoding any one the polypeptide and block polypeptide sequences as defined in the preceding paragraphs; an expression vector comprising said nucleic acid molecule; and a host cell comprising said nucleic acid molecule.
  • the invention provides a method of preparing a polypeptide, comprising: a) introducing the aforementioned expression vector into a host cell; b) culturing the host cells in a culture medium, under conditions allowing the expression of the polypeptide in said host cells and secretion of the polypeptide into the culture medium; c) removing said host cells from the culture medium, so as to provide a cell-free culture medium containing the secreted polypeptide by microfiltration, optionally preceded by centrifugation; d) optionally concentrating the polypeptide in the cell-free culture medium by ultrafiltration e) optionally removing low molecular weight components from the polypeptide and culture medium by ultrafiltration/diafiltration f) precipitating specifically the polypeptide from the cell-free culture medium by differential precipitation with a salt, preferably with ammonium sulfate, preferably at 20-80% of the saturating ammonium sulfate concentration, more preferably at 40-60% of the saturating
  • the invention provides a method of preparing a polypeptide comprising: a) introducing the aforementioned expression vector into a host cell; b) culturing the host cells in a culture medium, under conditions allowing the expression of the polypeptide in said host cells and secretion of the polypeptide into the extracellular culture medium; c) removing said host cells from the culture medium, so as to provide a cell-free culture medium containing the secreted polypeptide by microfiltration, optionally preceded by centrifugation; d) optionally concentrating the polypeptide in the cell-free culture medium by ultrafiltration e) optionally removing low molecular weight components from the polypeptide and culture medium by ultrafiltration/diafiltration f) purifying the polypeptide by chromatography, for example anion exchange chromatography g) removing salts and/or buffers from the polypeptide and optionally concentrating the polypeptide by ultrafiltration/diafiltration or dialysis, h) drying the polypeptide by spray drying
  • the invention provides a method of preparing a polypeptide, comprising: a) introducing the expression vector of claim 7 into host cells; b) culturing the host cells in a culture medium, under conditions allowing the expression of the polypeptide in said host cells; c) lysing the host cells resulting in a dispersion comprising the polypeptide and lysed cell debris; d) separating said debris from the dispersion, by a separation technique selected from the group consisting of centrifugation,, ultrafiltration, microfiltration, and combinations thereof; e) optionally removing low-molecular weight components from the polypeptide-containing dispersion by ultrafiltration or dialysis; f) purifying the polypeptide by a combination of differential precipitation and or chromatography; g) desalting the polypeptide obtained in step (d), (e) or (f) by ultrafiltration or dialysis and optionally drying the polypeptide.
  • Fig. 1 schematically shows a cloning procedure to generate a gene construct encoding a block co-polypeptide of the invention.
  • the invention generally relates to gellable polypeptides comprising at least two trimerizing blocks (“Tbiock’) and at least one not intrinsically trimerizing block (“Gbiock’), the alternating Tbiock and Gbiock modules in the polypeptide providing gelling capability.
  • Tbiock trimerizing blocks
  • Gbiock not intrinsically trimerizing block
  • the trimerizing blocks are trimer-forming oligopeptide blocks, the presence of which results in the polypeptides adopting a triple-helix conformation.
  • the invention is based on the judicious insight to thereby provide, for the Gbiock a sequence comprising one or more natural oligopeptide stretches found in different bovine type I alphal collagen sequences, i.e., any one or more of the aforementioned sequences II, III, IV, and V.
  • a novel polypeptide sequence is provided consisting of a combination of two or more of these natural oligopeptide stretches.
  • a further preferred novel polypeptide sequence is described, according to the one letter nomenclature for polypeptides as having the amino acid sequence according to Formula (I): (GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ)q (I) wherein q is an integer of from 1 to 40. Preferably q is 2 to 20, more preferably 3 to 10. Still more preferably q is 3-8, most preferably 5.
  • amino acid sequences, as defined herein amino acids are denoted by single-letter symbols. These single-letter symbols and three-letter symbols are well known to the person skilled in the art.
  • a (Ala) is alanine
  • D (Asp) is aspartic acid
  • E (Glu) is glutamic acid
  • G (Gly) is glycine
  • K (Lys) is lysine
  • N (Asn) is asparagine
  • P (Pro) is proline
  • Q (Gin) is glutamine
  • R (Arg) is arginine
  • S (Ser) is serine.
  • novel polypeptide comprises at least one sequence of a set of four natural oligopeptide stretches, each found in bovine type I alpha 1 collagen:
  • the polypeptide comprises any one or more of said oligopeptide stretches.
  • the polypeptide possibly comprises a single one of each of the sequences of the formulae II, III, IV, and V, whereby said single sequence possibly is present one or more times, such as up to 5 times.
  • the polypeptide possibly comprises two, three, or four of each of the sequences of the formulae II, III, IV, and V, irrespective of the number of occurrences of each such sequence, and the order in which these occur.
  • said sequences are present in the order of II-III-IV-V, i.e., GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ.
  • the maximum number of amino acid residues in the polypeptide is not specifically limited, but will preferably be below 6000, more preferably below 3000.
  • the number of amino acid residues in the polypeptide is in a range of from 30 to 1200, more preferably 50 to 1000.
  • the term “amino acid residue” as used in this disclosure has a known meaning. Accordingly, as the person skilled in the art of polypeptides will recognize, the term “amino acid residues” refers to polypeptide-incorporated amino acid molecules, i.e., to the amino acids when present in a peptide chain, Thereby the amino and carboxylic functional groups of the original amino acids present in said chain are engaged in peptide bonds.
  • the amino acid residues include the terminal amino acids of a peptide chain, in which either of said amino and carboxylic groups is engaged in a peptide bond, the other being an end-group of the peptide chain.
  • the polypeptide comprises all four of said sequences II to V, and more preferably the polypeptide satisfies the structure of Formula (I).
  • the aforementioned polypeptide with any of the sequences as described, and preferably being of formula (I), can be produced as such.
  • an intermediate is provided that can be used in producing a gellable polypeptide by chemically attaching trimerizing blocks to it.
  • the DNA in a suitable vector, is expressed in a host cell.
  • Suitable hosts are, e.g., Pichia pastoris, Hansenula polymorpha, Kluyveromyces marxianus var. Lactis, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus awamori, Bacillus megaterium, Bacillus brevis, Bacillus subtilis, Bacillus thuringiensis, E. coli K12 derivative.
  • the preferred host is Pichia pastoris and the preferred mode of expression is secretion into the extracellular medium.
  • the polypeptide of formula (I) is produced in the form of one or more blocks contained in an overall gellable polypeptide structure. It will be understood that, in such event, the terminal amino acids of the polypeptide of formula (I) will have a peptide bond linkage to an adjacent trimerizing block. Preferably, this is accomplished in such a way as to provide a polypeptide satisfying formula (Tblock-Gblock)n-Tblock (VI) wherein: n is an integer of from 1 to 18;
  • Gbiock represents the polypeptide defined hereinabove, having an amino acid sequence comprising at least one of each of said oligopeptides of formula (II), (III), (IV), and (V), more preferably the aforementioned peptide block of formula (I) satisfying the structure (GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ)q, wherein q is an integer of from 1 to 40, preferably 2 to 20, more preferably 3 to 10, still more preferably 3-8, and most preferably 5.
  • the total number of amino acid residues in (Tbiock-Gbiock)n-Tbiock preferably being at most 1500, more preferably at most 1200.
  • Tbiock has the peptide sequence (GPP)k, with k preferably being 7 to 16, more preferably 8 to 12.
  • the invention further encompasses nucleic acid constructs, including DNA and RNA molecules, that encode the polypeptides described herein. Accordingly, in one embodiment, triblock [(Tbiock- Gbiock)n-Tbiock] genes are constructed and transfected to an expression host, preferably P. pastoris, using a Pichia pastoris expression vector.
  • the invention also features vectors that include the present nucleic acid constructs.
  • expression vectors especially those for expression in eukaryotic cells.
  • Such vectors can, for example, be viral, plasmid, cosmid, or artificial chromosome (e.g., yeast artificial chromosome) vectors.
  • plasmids are circular, dsDNA elements that include one or more cloning sites for insertion of selected DNA sequences, e.g., coding sequences.
  • Such plasmids may include a functional origin of replication and thus are replication competent, or may be replication defective.
  • the corresponding yeast expression vectors are preferred, more preferably Pichia pastoris expression vectors, even more preferring Pichia pastoris expression vectors with a suitable signal sequence for secretory expression, most preferably Pichia pastoris expression vectors with a suitable pre-pro-sequence for efficient polypeptide secretion, and conferring stable integration into the genome of Pichia pastoris at a specifically targeted locus, after linearization of the expression vector with a suitable corresponding endonuclease and transfection of the linearized vector into the Pichia cells, for example by electroporation.
  • the present nucleic acid constructs can be introduced into the host cells growing in culture in vitro by conventional transfection techniques (e.g., calcium phosphate precipitation, DEAE-dextran transfection, electroporation, and other methods, preferably electroporation).
  • conventional transfection techniques e.g., calcium phosphate precipitation, DEAE-dextran transfection, electroporation, and other methods, preferably electroporation.
  • Another aspect of the invention pertains to host cells, preferably P. pastoris cells, into which a nucleic acid construct of the invention has been introduced, i.e., a “recombinant host cell”, preferably with a nucleic acid construct of the invention stably integrated into the genome of Pichia pastoris at a specifically targeted locus
  • polypeptides are thus produced in a method comprising: a) introducing an applicable expression vector, as described hereinbefore, into a host cell; b) culturing the host cells in a culture medium, under conditions allowing the expression of the polypeptide in said host cells; c) removing said host cells from the culture medium containing the secreted polypeptide by microfiltration, optionally preceded by centrifugation; d) optionally concentrating the polypeptide in the cell-free culture medium by ultrafiltration e) optionally removing low molecular weight components from the polypeptide and culture medium by ultrafiltration/diafiltration f) precipitating specifically the polypeptide from the cell-free culture medium by differential precipitation with a salt, preferably with ammonium sulfate, preferably at 20-80% of the saturating ammonium sulfate concentration, more preferably at 40-60% of the saturating ammonium sulfate concentration; g) dissolving the precipitated polypeptide in water; h
  • the invention provides a method of preparing a polypeptide comprising: a) introducing the aforementioned expression vector into a host cell; b) culturing the host cells in a culture medium, under conditions allowing the expression of the polypeptide in said host cells and secretion of the polypeptide into the extracellular culture medium; removing said host cells from the culture medium, so as to provide a cell-free culture medium containing the secreted polypeptide by microfiltration, optionally preceded by centrifugation; d) optionally concentrating the polypeptide in the cell-free culture medium by ultrafiltration e) optionally removing low molecular weight components from the polypeptide and culture medium by ultrafiltration/diafiltration f) purifying the polypeptide by chromatography, for example anion exchange chromatography g) removing the salts from the polypeptide and optionally concentrating the polypeptide by ultrafiltration/diafiltration or dialysis, h) drying the polypeptide by spray drying or freeze-drying.
  • the invention provides a method of preparing a polypeptide, comprising: a) introducing the aforementioned expression vector into a host cell; b) culturing the host cells in a culture medium, under conditions allowing the expression of the polypeptide in said host cells; c) lysing the host cells; d) separating the lysed cells, e) solubilizing the lysed cells to provide a solution, f) dialyzing the solution obtained in step (e), thereby obtaining isolated polypeptides g) drying the polypeptide.
  • the block co-polypeptide of the invention will generally be obtained in the form of a lyophilized or spray dried polypeptide.
  • the block co-polypeptide of the invention is capable of reversibly forming hydrogels upon cooling heated aqueous solutions of the polypeptide.
  • the temperature at which a gel is formed can be tuned by varying the length of the triple-helix forming blocks, i.e., the integers k, m., and p referred to above. Generally this temperature will vary from 10 to 80 °C, preferably from 15 to 50 °C, more preferably from 30-40 °C.
  • an aspect of the invention includes the use of said block-co-polypeptide as an ingredient capable of gelling in food and beverage applications.
  • desserts more particularly for gel formation, and/or for texture, transparency, and brilliance
  • fruit gummies more particularly for gel formation and/or for texture, elasticity, transparency and/or brilliance
  • marshmallows more particularly for foam formation, and/or for foam stabilization or gel formation
  • pastilles more particularly as a binding agent and/or for texture or melting properties, and to prevent disintegration
  • caramels more particularly as an emulsifier and foam stabilizer, and/or for chewability
  • yogurt more particularly for stabilization of syneresis, and/or for texture and creaminess
  • meat and sausages more particularly for emulsion stabilization and/or water/juice binding
  • broths and canned meats more particularly as a binding agent, and/or for texture as well
  • the block co-polypeptides of the present invention serve to better address the desire for providing an animal-free gelatin replacement, satisfying desired gelatin properties enabling its use in food applications.
  • this refers to functional properties such as: clarity, elastic texture, melt in mouth, for ready to eat (RTE) dessert gels; elastic texture, clarity, low hot viscosity, low set temperature in high solids confectionery; as a whipping/aeration agent, foam stabilizer, elastic texture in foamed confectionery - marshmallows; for elastic gel texture, fatlike mouthfeel, emulsion stabilization in low-fat spreads; for a creamy mouthfeel in stirred yogurt, and prevention of syneresis; as a whipping agent in desserts and mousses, and also for a creamy consistency and providing a low set temperature; for providing a smooth texture and a creamy mouthfeel to sour cream; and for improving and stabilizing the soft texture of ice cream.
  • functional properties such as: clarity, elastic texture, melt in mouth, for ready to eat (RTE) dessert
  • block co-polypeptides of the present invention are suitable for use in various cosmetic or medical application. These include a use as:
  • capsules for pharmaceutical formulations i.e., capsules for the administration of substances, such as drug substances;
  • polypeptide will be generally used in the form of a hydrogel.
  • the polypeptides of the invention can also be used, either in gel or in dry form, as a surgical aid to prevent post-operative sticking of tissues or organs to each other.
  • Another such use either in gel or in dry form as an aid in surgery to control blood flow, such as temporary control of blood flow during or after surgery.
  • a gelatin-replacement polypeptide that can be suitably produced in Pichia pastoris.
  • the polypeptide has a structure (Tbiock-Gbiock)n-Tbiock, wherein n is an integer of from 1 to 18.
  • Gbiock comprises any one or more of the oligopeptides having the amino acid sequences GPKGEPGSPGEN, GPKGNSGEP, GPSGEPGKQGPS, and GSPGEQ.
  • Gbiock has the amino acid sequence
  • (B) Gbiock consisting of a stretch of the natural bovine collagen type III (alpha 1) sequence.
  • each Gbiock block was fused with a Tbiock consisting of a nonablock triplet oligopeptide having the sequence (GPP)g.
  • Example 1 Three triblock gelatin-encoding genes, corresponding to (A), (B), and (C) of Example 1 were constructed and cloned into a Pichia pastoris vector. The three vectors were used to transform Pichia. Subsequently, benchtop methanol fed-batch fermentations were performed with selected Pichia strains. After cell removal by centrifugation and microfiltration, the proteins were purified from the cell-free broth by differential ammonium sulfate precipitation, and they were desalted by dialysis. For analytical purposes such as SDS-PAGE and mass spectrometry, the purification is normally done at (sub)-mL scale.
  • the purified proteins were subjected to polyacrylamide gel electrophoresis after treatment with SDS to denature the proteins and provide them with negative charges (SDS-PAGE). Therein polypeptide (C) showed only very minor degradation (note that the gel is overloaded). Polypeptides (A) and (B) showed some degradation, although still much less than typically seen for natural collagen sequences.
  • the three triblock proteins were subjected to MALDI-TOF to analyze their molecular mass distribution. This confirmed the conclusion from SDS-PAGE that proteolytic degradation is negligible in (0), while some level of degradation is visible in (A) and particularly in (B). In, addition, MALDI-TOF showed that uncharacterized post-translational modifications had occurred particularly in (A).
  • Apolypeptide (D) was made, as a nonablock variant of (0) of Example 1, i.e., with the integer n being 4.
  • a corresponding gene was constructed, encoding a 915 aa (83 kDa) protein with a calculated isoelectric point of 4.63.
  • the cloning procedure used to generate the nonablock constructs is shown in Figure 1.
  • the sequence of the construct was verified by Sanger sequencing from both ends, and by Nanopore long read sequencing to exclude the hypothetical possibility of rearrangements in the middle section (this cannot normally be verified by Sanger sequencing because of the long repetitive sequence).
  • the vector was then used to transform P. pastoris.
  • the nonablock gelatin (D) was successfully produced.

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Abstract

L'invention concerne un polypeptide de remplacement de gélatine qui peut être produit de manière appropriée dans Pichia pastoris. Le polypeptide a une structure (Tbloc-Gbloc)n-Tbloc, où n est un nombre entier de 1 à 18. Tbloc est, par exemple, (GPP)k, k étant compris entre 6 et 20. Gbloc comprend un ou plusieurs des oligopeptides ayant les séquences d'acides aminés GPKGEPGSPGEN, GPKGNSGEP, GPSGEPGKQGPS, et GSPGEQ, de préférence Gbloc a la séquence d'acides aminés (GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ)q, q étant de 1 à 40.
PCT/NL2024/050170 2023-04-06 2024-04-05 Polypeptides de remplacement de la gélatine Pending WO2024210747A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP23167140.5 2023-04-06
EP23167140.5A EP4442697A1 (fr) 2023-04-06 2023-04-06 Polypeptides de remplacement de gélatine
EP23181277.7 2023-06-23
EP23181277 2023-06-23

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009151327A1 (fr) 2008-06-11 2009-12-17 Stichting Dienst Landbouwkundig Onderzoek Co-polypeptide en blocs et hydrogels fabriqués à partir de celui-ci
WO2010079076A2 (fr) * 2008-12-17 2010-07-15 Mosaiques Diagnostics And Therapeutics Ag Maladie polykystique rénale autosomique dominante (adpkd)
WO2010091251A2 (fr) * 2009-02-06 2010-08-12 The University Of Medicine And Dentistry Of New Jersey Produits modulaires de type collagène à triple hélice
EP3315145A1 (fr) * 2016-10-28 2018-05-02 BSN medical GmbH Produit de parage multicouche avec couche de collagène perforée
CN114874316A (zh) * 2022-06-24 2022-08-09 重庆大学 一种水溶性胶原蛋白及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009151327A1 (fr) 2008-06-11 2009-12-17 Stichting Dienst Landbouwkundig Onderzoek Co-polypeptide en blocs et hydrogels fabriqués à partir de celui-ci
WO2010079076A2 (fr) * 2008-12-17 2010-07-15 Mosaiques Diagnostics And Therapeutics Ag Maladie polykystique rénale autosomique dominante (adpkd)
WO2010091251A2 (fr) * 2009-02-06 2010-08-12 The University Of Medicine And Dentistry Of New Jersey Produits modulaires de type collagène à triple hélice
EP3315145A1 (fr) * 2016-10-28 2018-05-02 BSN medical GmbH Produit de parage multicouche avec couche de collagène perforée
CN114874316A (zh) * 2022-06-24 2022-08-09 重庆大学 一种水溶性胶原蛋白及其应用

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
DATABASE UniProt [online] 21 July 1986 (1986-07-21), "Collagen alpha-1(I) chain precursor", XP002811733, retrieved from EBI accession no. UNIPROT:P02452 Database accession no. P02452 *
GOEDEN-WOOD N.L.CONTICELLO V.P.MULLER S.J.KEASLING J.D., BIOMACROMOLECULES, vol. 3, 2002, pages 874 - 879
HENDERSON D.B.DAVIS R.M.DUCKER W.A.VAN COTT K.E., BIOMACROMOLECULES, vol. 6, 2005, pages 1912 - 1920
LU Q., TRENDS IN BIOTECHNOL., vol. 23, no. 4, 2005, pages 199 - 207
MCMILLAN R.A.LEE T.A.T.CONTICELLO V.P., MACROMOLECULES, vol. 32, 1999, pages 3643 - 3648
MI L., BIOMACROMOLECULES, vol. 7, 2006, pages 2099 - 2107
PADGETT K.A.,SORGE J.A., GENE, vol. 168, 1996, pages 31 - 35
WERTEN ET AL., BIOMACROMOLECULES, vol. 10, 2009, pages 1106 - 1113
WERTEN ET AL., YEAST, vol. 15, no. 11, pages 1087 - 1096
WERTEN M W T ET AL: "Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris", PROTEIN ENGINEERING, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 14, no. 6, 1 June 2001 (2001-06-01), pages 447 - 454, XP002302730, ISSN: 0269-2139, DOI: 10.1093/PROTEIN/14.6.447 *
WERTEN M.W.T.WISSELINK W.H.JANSEN-VAN DEN BOSCH T.J.BRUIN E.C.WOLF F.A., PROTEIN ENGINEERING, vol. 14, no. 6, 2001, pages 447 - 454
WON J.-I.BARRON A.E., MACROMOLECULES, vol. 35, 2002, pages 8281 - 8287

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