WO2024210747A1

WO2024210747A1 - Gelatin-replacement polypeptides

Info

Publication number: WO2024210747A1
Application number: PCT/NL2024/050170
Authority: WO
Inventors: Marc Willem Theodoor Werten; Zheng YOU
Original assignee: Stichting Wageningen Research; Tate & Lyle Solutions Usa Llc
Priority date: 2023-04-06
Filing date: 2024-04-05
Publication date: 2024-10-10

Abstract

Disclosed is a gelatin-replacement polypeptide that can be suitably produced in Pichia pastoris. The polypeptide has a structure (T_block-G_block)_n-T_block, wherein n is an integer of from 1 to 18. T_block is, for example, (GPP)_k, with k being 6-20. G_block comprises any one or more of the oligopeptides having the amino acid sequences GPKGEPGSPGEN, GPKGNSGEP, GPSGEPGKQGPS, and GSPGEQ, preferably G_block has the amino acid sequence (GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ)q with q being 1 to 40.

Description

Title: GELATIN-REPLACEMENT POLYPEPTIDES

Field

The invention is in the field of gelatin-replacement polypeptides. Particularly, the invention pertains to tri- and multi-block polypeptides capable of being produced in yeast host cells, and capable of gelling.

Background

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. These crosslinks must be largely hydrolyzed in order to make the collagen and - after heat denaturation - the resulting gelatin, soluble. However, 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.

As a further drawback of natural gelatin, inter alia 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. In food, and even more strongly in pharmaceutical applications, this is a critical issue, which has resulted in regulatory restrictions on the origin of the gelatins used in these products. Moreover, a tendency in society is to try and reduce cattle breeding in view of environmental concerns. As a result, 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. Interestingly, yeast -derived, fungal or bacterial products can be considered to be suitable replacements as being, inter alia, vegan as well as kosher or halal.

Thus, it is desirable to be able to avoid animal collagen as a source for gelatin. To this end 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. Further background literature on the production of custom-designed gelatins in Pichia pastoris is Werten et al., Protein Engineering (2001), vol.14, no.6, pp. 447-454. All of these references are directed at avoiding animal polypeptide sources, and advocating custom-designed gelatins.

Producing artificial, mammahan (e.g., bovine or porcine), designed polypeptide, also comes with a disadvantage. On the one hand, it is desired to avoid animal gelatin. On the other hand, the public at large prefers natural products over artificial products, particularly with respect to food. In view hereof, it is preferred to step away from fully artificial peptide sequences, and build a polypeptide made up of stretches of natural sequences.

The foregoing artificial polypeptides are produced as extracellular proteins in recombinant yeast, specifically in transformed Pichia pastoris. Background art on such production in Pichia pastoris can be found in Werten et al., Yeast 15(11): 1087-1096. In view of, inter alia, its GRAS status for heterologous protein production, 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. This is particularly true for unfolded proteins such as gelatins. Also the gel forming ability of gelatin-like polypeptides produced in Pichia is not normally achieved. Natural collagen sequences, when produced by Pichia, do not have the aforementioned triple helix-forming and gel-forming capacity. This is because, in contrast to animal collagen-producing cells, Pichia does not naturally carry out the post-translational modifications required for this capacity.

It is thus desired to provide 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.

Summary of the invention

In order to better address one or more of the above desires, 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)

GPSGEPGKQGPS (IV)

GSPGEQ (V), 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.

In another aspect, 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;

- Tbiock, each independently denotes a trimerizing peptide block selected from the group consisting of (GPP)k, (GER) _m, (GRE) _m, (GEK)_m, (GKE)m, (GPKGDP)p, and (GPKGEP)_p, wherein k is an integer from 6 to 20, preferably 7 to 16, more preferably 8 to 12, m is an integer of from 12 to 20 and p is an integer of from 3 to 12;

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.

In a further aspect, the invention provides a hydrogel comprising a block co-polypeptide as defined in the preceding paragraph.

In still further aspects, 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.

In yet another aspect, 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 ammonium sulfate concentration; g) dissolving the precipitated polypeptide in water; h) optionally repeating the above precipitation and dissolution of the polypeptide once, to increase the purity of the polypeptide; i) removing the salt from the polypeptide and optionally concentrating the polypeptide by ultrafiltration/diafiltration or dialysis, j) drying the polypeptide by spray drying or freeze-drying.

In yet another aspect, 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 or freeze-drying.

In yet another aspect, 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.

Drawings

Fig. 1 schematically shows a cloning procedure to generate a gene construct encoding a block co-polypeptide of the invention.

Detailed description

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. As the skilled person will understand, the trimerizing blocks are trimer-forming oligopeptide blocks, the presence of which results in the polypeptides adopting a triple-helix conformation.

In a broad sense, 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. Preferably 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. In 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. For completeness’ sake, we note that for the amino acids referred to in the present disclosure the single-letter symbols have the following meaning: 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, and S (Ser) is serine.

The novel polypeptide comprises at least one sequence of a set of four natural oligopeptide stretches, each found in bovine type I alpha 1 collagen:

GPKGEPGSPGEN (II)

GPKGNSGEP (III)

GPSGEPGKQGPS (IV)

GSPGEQ (V)

Hereby the polypeptide comprises any one or more of said oligopeptide stretches. Accordingly, 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. Possibly, 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. Preferably, 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, including repetitions of the aforementioned sequences, is not specifically limited, but will preferably be below 6000, more preferably below 3000. Preferably, 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.

Preferably, the polypeptide comprises all four of said sequences II to V, and more preferably the polypeptide satisfies the structure of Formula (I).

Optionally, the aforementioned polypeptide, with any of the sequences as described, and preferably being of formula (I), can be produced as such. In that event, an intermediate is provided that can be used in producing a gellable polypeptide by chemically attaching trimerizing blocks to it. Although this is technically possible, those skilled in the art will appreciate that producing the entire polypeptide by means of recombinant technologies is preferred. This better allows to produce long and monodisperse polypeptides with a defined sequence order.

Polypeptide manufacture through recombinant technologies is known. In general an artificial DNA sequence is provided in a known manner, having a sequence encoding for the desired polypeptide. Various cloning methods are nowadays available for the construction of G-C-rich, repetitive DNA sequences and genes of interest (Padgett K.A. & Sorge J.A. (1996) Gene 168: 31-35; McMillan R.A., Lee T.A.T. & Conticello V.P. (1999) Macromolecules 32: 3643-3648; Werten M.W.T., Wisselink W.H., Jansen-van den Bosch T.J., de Bruin E.C. & de Wolf F. A. (2001) Protein Engineering 14(6): 447-454; Goeden-Wood N.L., Conticello V.P., Muller S.J. & Keasling J.D. (2002) Biomacromolecules 3: 874-879; Won J. -I. & Barron A.E. (2002) Macromolecules 35: 8281-8287; Henderson D.B., Davis R.M., Ducker W.A., Van Cott K.E. (2005) Biomacromolecules 6: 1912-1920; Lu Q. (2005) Trends in Biotechnol. 23(4): 199-207; Mi L. (2006) Biomacromolecules 7: 2099-2107). This does not require elucidation here. 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.

Thus, preferably, 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;

- Tbiock, each independently denotes a trimerizing peptide block selected from the group consisting of (GPP)k, (GER) _m, (GRE) _m, (GEK)_m, (GKE) m, (GPKGDP) _p, and (GPKGEP) _p, wherein k is an integer from 6 to 20, preferably 7 to 16, more preferably 8 to 12 m is an integer of from 12 to 20 and p is an integer of from 3 to 12;

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.

In a preferred embodiment, 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. Of particular benefit are 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. Typically, 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. In view of the preferred production in yeast, particularly in Pichia pastoris, it will be understood that 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.

With reference to the possible repetition of the various polypeptide sequences, i.e., the aforementioned integers k, n, m, and p, corresponding cloning procedures can be carried out. Such procedures as such are well-known in the art. Reference is also made to Figure 1, which shows a cloning procedure to generate the gene constructs encoding (Tbiock-Gbiock)n-Tbiock as well as the subsequent transformation of P. pastoris.

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).

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

Overall, 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) optionally repeating the above precipitation and dissolution of the polypeptide once, if needed to increase the purity of the polypeptide; i) removing the salts from the polypeptide and optionally concentrating the polypeptide by ultrafiltration/diafiltration or dialysis, j) drying the polypeptide by spray drying or freeze-drying.

In yet another aspect, 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.

In yet another aspect, 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.

Just as natural gelatin, 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.

The block co-polypeptide of the invention presented in this disclosure is suitable as a substitute for gelatin in foods and beverages. Accordingly, an aspect of the invention includes the use of said block-co-polypeptide as an ingredient capable of gelling in food and beverage applications. Particularly this concerns 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), and broths and canned meats (more particularly as a binding agent, and/or for texture as well as sliceability of canned meats).

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. In addition to the foregoing, 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.

In interesting further embodiments, the block co-polypeptides of the present invention are suitable for use in various cosmetic or medical application. These include a use as:

- a rheology modifier in cosmetics or personal care formulations;

- a skin-restoring agent in cosmetics or personal care formulations;

- a material for the production of capsules for pharmaceutical formulations, i.e., capsules for the administration of substances, such as drug substances;

- a material for the production of gels for the controlled and/or extended release of substances, such as drug substances;

- a scaffold material for tissue culture or tissue engineering;

- an additive to promote the stability, viability and/or shelflife of cells, tissues, vaccines.

In these applications, the 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.

In sum, a gelatin-replacement polypeptide is disclosed 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. Preferably Gbiock has the amino acid sequence

(GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ)q wherein q is an integer of from 1 to 40.

The invention will be illustrated hereinafter with reference to the following non-limiting Examples.

Example 1

Three gelatin designs were selected, each of a general structure (Tblock-Gblock)n-Tblock with n — 1.

(A) Gbiock consisting of a stretch of the the natural bovine collagen type I (alpha 1) sequence.

(B) Gbiock consisting of a stretch of the the natural bovine collagen type III (alpha 1) sequence.

(C) Gbiock consisting of the polypeptide of Formula (I).

In all three polypeptides the N- and C-terminal ends of each Gbiock block were fused with a Tbiock consisting of a nonablock triplet oligopeptide having the sequence (GPP)g.

(A)

• 255 amino acids

• molecular weight: 22.52 kDa

• IEP: 8.34

• Amino acid sequence:

(GPP)₉-GPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPG AEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRG ETGPAGPAGPIGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGL QGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGSPGKDGLNGLPGPIGPP

GPRGRTGDAGPA-(GPP)₉

(B)

• 249 amino acids

• molecular weight: 21.59 kDa

• IEP: 5.20

• Amino acid sequence:

(GPP)₉-GERGGPGGPGPQGPAGKNGETGPQGPPGPTGPSGDKGDTGPPG PQGLQGLPGTSGPPGENGKPGEPGPKGEAGAPGIPGGKGDSGAPGERG PPGAGGPPGPRGGAGPPGPEGGKGAAGPPGPPGSAGTPGLQGMPGERG GPGGPGPKGDKGEPGSSGVDGAPGKDGPRGPTGPIGPPGPAGQPGDKG

ESGAPGVP-(GPP)9

(C)

• 249 amino acids

• molecular weight: 22.37 kDa

• IEP: 4.63

• Amino acid sequence:

(GPP)₉-GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQGPKG EPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQGPKGEPGSPGENG PKGNSGEPGPSGEPGKQGPSGSPGEQGPKGEPGSPGENGPKGNSGEPG PSGEPGKQGPSGSPGEQGPKGEPGSPGENGPKGNSGEPGPSGEPGKQG

PSGSPGEQ-(GPP)9

Example 2

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).

Gel formation was qualitatively assessed for the (A), (B), and (0) triblock gelatins. To this end, solutions of the three purified proteins in water at 10% (w/v) were prepared and heated for 5 min. at 45 °C to melt any triple helices. The gels were then allowed to cool to room temperature. Gel formation within 5 min. was observed only for (0), as judged by the conventional “inverted tube test”. After overnight incubation all triblocks formed gels.

Example 3

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. (D)

• 915 amino acids

• molecular weight: 82.64 kDa

• IEP: 4.63

• Amino acid sequence:

(GPP)₉-GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQGPKG EPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQGPKGEPGSPGENG PKGNSGEPGPSGEPGKQGPSGSPGEQGPKGEPGSPGENGPKGNSGEPG PSGEPGKQGPSGSPGEQGPKGEPGSPGENGPKGNSGEPGPSGEPGKQG PSGSPGEQ-(GPP)₉-GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGS PGEQGPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQGPKGE PGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQGPKGEPGSPGENGP KGNSGEPGPSGEPGKQGPSGSPGEQGPKGEPGSPGENGPKGNSGEPGP SGEPGKQGPSGSPGEQ-(GPP)₉-GPKGEPGSPGENGPKGNSGEPGPSGEP GKQGPSGSPGEQGPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSP GEQGPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQGPKGEP GSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQGPKGEPGSPGENGPK GNSGEPGPSGEPGKQGPSGSPGEQ-(GPP)9- GPKGEPGSPGENGPKGNS GEPGPSGEPGKQGPSGSPGEQGPKGEPGSPGENGPKGNSGEPGPSGEP GKQGPSGSPGEQGPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSP

GEQGPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQGPKGEP GSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ-(GPP)9

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.

Claims

1. A polypeptide comprising at least one oligopeptide selected from the group consisting of the oligopeptides of formula (II), (III), (IV), and (V):

GPKGEPGSPGEN (II)

GPKGNSGEP (III)

GPSGEPGKQGPS (IV)

GSPGEQ (V).

2. The polypeptide of claim 1, having an amino acid sequence comprising at least one of each of the oligopeptides of formula (II), (III), (IV), and (V).

3. The polypeptide of claim 2, having the amino acid sequence (GPKGEPGSPGENGPKGNSGEPGPSGEPGKQGPSGSPGEQ)q wherein q is an integer of from 1 to 40, preferably 3 to 10.

4. A block co-polypeptide having a structure (Tbiock-Gbioc k)n"Tblock, wherein n is an integer of from 1 to 18; Tbiock denoted, each independently, a trimerizing peptide block having an amino acid sequence selected from the group consisting of (GPP)k, (GER)_m, (GRE)_m, (GEK)_m, (GKE)_m, (GPKGDP)_p, and (GPKGEP)p, wherein k is an integer from 6 to 20, preferably 7 to 16, more preferably 8 to 12, m is an integer of from 12 to 20 and p is an integer of from 3 to 12; and Gbiock represents a polypeptide as defined in any one of the claims 1 to 3.

5. The block co-polypeptide of claim 4, wherein the total number of amino acid residues in (Tbiock-Gbiock)n-Tbiock is at most 1500, preferably at most 1200.

6. The block co-polypeptide of claim 4 or 5, wherein the Tbiock modules are (GPP)k, and k is 8 to 12, preferably wherein Gbiock represents the polypeptide of claim 2.

7. The block co-polypeptide of claim 6, wherein Gbiock represents the polypeptide of claim 3.

8. A hydrogel comprising a block co-polypeptide as defined in any one of the claims 4 to 7.

9. A nucleic acid construct encoding the block co-polypeptide as defined in any one of the claims 4 to 7.

10. An expression vector comprising the nucleic acid construct of claim 9.

11. The expression vector of claim 10, wherein the vector is a Pichia pastoris expression vector.

12. A host cell comprising the expression vector of claim 11.

13. The host cell of claim 12, being a Pichia pastoris host cell comprising the expression vector of claim 11.

14. A method of preparing a polypeptide, comprising: a) introducing the expression vector of claim 10 into host cells; 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 said 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 ammonium sulfate concentration; g) dissolving the precipitated polypeptide in water; h) optionally repeating the above precipitation and dissolution of the polypeptide; i) removing the salt from the polypeptide and optionally concentrating the polypeptide by ultrafiltration/diafiltration or dialysis, j) optionally drying the polypeptide by spray drying or freeze-drying.

15. A method of preparing a polypeptide, comprising: a) introducing the expression vector of claim 10 into host cells; 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 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) optionally drying the polypeptide by spray drying or freeze-drying.

16. A method of preparing a polypeptide, comprising: a) introducing the expression vector of claim 10 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.

17. The method of claim 14, 15, or 16, wherein the expression vector is as defined in claim 11, and wherein the host cells are Pichia pastoris host cells.

18. Use of a block-co-polypeptide according to any one of the claims 4 to 7 as an ingredient capable of gelling in food and beverage applications, particularly selected from the group consisting of desserts, fruit gummies, marshmallows, pastilles caramels yogurt, meat, sausages, broths, and canned meats.

19. Use of a block-co-polypeptide according to any one of the claims 4 to 7 as a rheology modifier in cosmetics or personal care formulations.

20. Use of a block-co-polypeptide according to any one of the claims 4 to 7 as a skin-restoring agent in cosmetics or personal care formulations.

21. Use of a block-co-polypeptide according to any one of the claims 4 to 7 for the production of capsules for pharmaceutical formulations.

22. Use of a block-co-polypeptide according to any one of the claims 4 to 7 for the production of gels for the controlled and/or extended release of substances.

23. Use of a block-co-polypeptide according to any one of the claims 4 to 7 as a scaffold material for tissue culture or tissue engineering.

24 Use of a block-co-polypeptide according to any one of the claims 4 to 7, as an additive to promote the stability, viability and/or shelflife of cells, tissues, or vaccines.

25. Use of a block-co-polypeptide according to any one of the claims 4 to 7, either in gel or in dry form, as a surgical aid to prevent post-operative sticking of tissues or organs to each other.

26. Use of a block-co-polypeptide according to any one of the claims 4 to 7, either in gel or in dry form as an aid in surgery to control blood flow.