Patent Application
20250002721
The present invention relates to novel carbohydrate binding polypeptides and their use in making hydrogels.
Hydrogels are a huge and booming business, with application in many fields including drug delivery, tissue scaffolding, life science research, the cosmetic industry, bio-based agriculture, and industrial lubrication. The majority of hydrogels are still made from fossil-derived polymers, built into a 3D network by chemical modification and cross-linking. This production method is unsustainable, and it is viewed unfavourably by consumers, so there is a pressing need to start using biological polymers for gel formation.
Hydrogels can already be produced from some natural polymers like cellulose and other polysaccharides of plant or microbial origin, as well as animal-derived mucins. But these still require chemical modification to permit cross-linking, leading to a high chemical waste footprint, and the potential inclusion of compounds that can irritate the skin or interfere with other biological systems, depending on the application.
There is a strong drive from industry to develop new methods of producing hydrogels from sustainably sourced polysaccharides such as cellulose and scleroglucan. Scleroglucan is secreted by industrial fungi such as Sclerotium rolfsii, and can be obtained with reasonably high yield and purity (Schmid et al. 2011). Prior to gel formation, in current practice scleroglucan is typically dissolved in DMSO in order to separate the triple helices of its structure (Palleschi et al. 2005). It is then oxidised using sodium periodate to generate scleraldehyde derivatives (Maeda et al. 2001) with in some cases further oxidation catalysed by sodium chlorite, which produces carboxylic groups, yielding a product termed sclerox (Crescenzi et al. 1983). This oxidised form can be cross-linked with 1,6-hexanedibromide to create a gel-forming network, and different sclerox:dihalide ratios give markedly different mechanical and rheological properties (Coviello et al. 1999; Coviello et al. 2001). More recently, an efficient alternative method was developed that uses boric acid (Borax) to directly cross-link the polysaccharide by means of 4,6-gluco-borate linkages and physical associations, creating a physical gel (Coviello et al. 2005; Palleschi et al. 2006). However, Borax is a regulated Substance of Very High Concern (SVHC) (European Chemicals Agency).
Thus, the need for improving sustainability of hydrogel production remains unsolved.
We here present an entirely new mode of polysaccharide cross-linking that requires no modification of the starting polymer and that uses no chemical cross-linking. The cross-linker is in fact a polypeptide belonging to a novel Carbohydrate Binding Module (CBM) family. Each polypeptide within the herein disclosed new CBM family, called CBM92, comprises three distinct repeat regions which are binding sites for mono-, oligo-, and polysaccharides. The polypeptides of this novel CBM family have binding specificity for polysaccharides with high molecular weight, e.g. scleroglucan, and gelation tendencies. The present inventors have demonstrated that proteins of this novel CBM family are highly advantageous as cross-linkers in hydrogel formation.
One aspect of the present disclosure relates to a polypeptide comprising at least two repeat regions selected from the group consisting of:
Y
V
12
13
W
E
F
35
36
38
Y
V
11
12
W
E
F
32
33
35
W
E
F
36
37
39
Another aspect of the present disclosure relates to a polypeptide comprising at least two repeat regions selected from the group consisting of:
A further aspect of the present disclosure relates to an amino acid sequence having at least 75% sequence identity to SEQ ID NO:3 or SEQ ID NO:4, or any of the sequences SEQ ID NO:20 to SEQ ID NO:33, or any of the sequences SEQ ID NO:71 to SEQ ID NO: 219, and wherein said polypeptide is capable of binding one or more saccharide units.
A further aspect of the present disclosure relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20 to SEQ ID NO:33, and wherein said polypeptide is capable of binding one or more saccharide units.
A further aspect of the present disclosure relates to a polynucleotide encoding a polypeptide disclosed herein.
Another aspect of the present disclosure relates to a recombinant vector comprising a polynucleotide disclosed herein, or a nucleic acid sequence encoding a carbohydrate-binding polypeptide disclosed herein.
Another aspect of the present disclosure relates to a host cell expressing the recombinant vector disclosed herein.
Another aspect of the present disclosure relates to a use of at least one polypeptide disclosed herein for cross-linking two or more polysaccharides.
A further aspect of the present disclosure relates to a hydrogel comprising at least one polypeptide disclosed herein and two or more polysaccharides, wherein said hydrogel has a cross-linked structure.
A further aspect of the present disclosure relates to a cross-linker for crosslinking polysaccharides, wherein said cross-linker is a polypeptide as disclosed herein.
Another aspect of the present disclosure relates to a method of manufacturing a hydrogel, the method comprising
As used herein, the term “polypeptide” refers to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. As the term does not refer to a specific length of the product, peptides, oligopeptides, and proteins are included within the definition of polypeptide. In an embodiment, the term “polypeptides” as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the disclosure as described herein. Amino acid sequence mutants of the polypeptides of the present disclosure can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the present invention, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
An “amino acid residue” can be a natural or non-natural amino acid residue linked by peptide bonds or bonds different from peptide bonds. The amino acid residues can be in D-configuration or L-configuration. An amino acid residue comprises an amino terminal part (NH2) and a carboxyl terminal part (COOH) separated by a central part comprising a carbon atom, or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group. NH2 refers to the amino group present at the amino terminal end of an amino acid or peptide, and COOH refers to the carboxyl group present at the carboxyl terminal end of an amino acid or peptide. The generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids of standard nomenclature as listed in J. Biol. Chem., 243:3552-59 (1969) and adopted in 37 C.F.R., section 1.822 (b)(2) belong to the group of amino acids listed herewith: Y, G, F, M, A, S, I, L, T, V, P, K, H, Q, E, W, R, D, N and C. Non-natural amino acids are those not listed immediately above. Also, non-natural amino acid residues include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues.
A “functional variant” of a peptide is a peptide capable of performing essentially the same functions as the peptide it is a functional variant of. In particular, a functional variant can bind the same molecules, preferably with the same affinity, as the peptide it is a functional variant of.
The terms “polynucleotide” and “nucleic acid” are used interchangeably herein, and can refer to any nucleic acid that contains the information necessary for the purpose indicated by the context. That is, the nucleic acid can be DNA or RNA, either single stranded or double stranded, or other nucleic acid, as long as it is capable of representing the appropriate information, e.g., in relation to an encoded peptide, and can include complementary sequences, e.g., sense strands and anti-sense strands of nucleic acids polymers.
As used herein, the term “binding site” refers to a region of a molecule or molecular complex that, as a result of its shape, favourably associates with another molecule, molecular complex, chemical entity or compound. The polypeptide of the disclosure described herein contains carbohydrate binding sites, meaning binding sites capable of associating with sugars, e.g. monosaccharides, disaccharides, and polysaccharides.
As used herein, the term “hydrogel” or “hydrophilic gel” refers to a continuous phase of a hydrophilic polymer that is capable of swelling on contact with water and other hydrophilic swelling agents. The term is used regardless of the state of hydration. A “hydrogel” refers to a material of solid or semi-solid texture that comprises water. Hydrogels are formed by a three-dimensional network of molecular structures within which water, among other substances, may be held. The three-dimensional molecular network may be held together by covalent chemical bonds, or by ionic bonds, or by any combination thereof.
“Polysaccharide”, “carbohydrate” or “oligosaccharide”: The terms “polysaccharide”, “carbohydrate”, or “oligosaccharide” refer to a polymer of sugars. The terms “polysaccharide”, “carbohydrate”, and “oligosaccharide”, may be used interchangeably. Typically, a polysaccharide or oligosaccharide comprises at least three sugars. The polymer may include natural sugars (e.g., glucose, fructose, galactose, mannose, arabinose, ribose, and xylose) and/or modified sugars (e.g., 2′-fluororibose, 2′-deoxyribose, and hexose). The term “saccharide” is used herein as a generic term for polymers of sugars and may refer to “monosaccharide”, “disaccharide”, “polysaccharide”, “carbohydrate”, or “oligosaccharide” and other synonyms.
A carbohydrate binding module (CBM) is a low molecular weight, non-catalytic protein that can bind to a specific carbohydrate ligand. CBMs are classified by sequence homology into families on the CAZy database (www.cazy.org) (Lombard et al. 2014). The mode of CBM-ligand binding is determined by the surface architecture of the CBM protein structure (Boraston et al. 2004). In most CBM families, the proteins present one carbohydrate binding site per protein.
One aspect of the present disclosure relates to novel polypeptides which belong to members of the CBM family 92 (CBM92). In one aspect, the novel polypeptides of the present disclosure are capable of binding at least two polysaccharides, preferably three polysaccharides, and cross-link them. The obtained cross-linked structure comprising the novel polypeptides of the present disclosure and two or three polysaccharides, in presence of water, forms a hydrogel. Thus, in one aspect of the present disclosure, the novel polypeptides are characterized by comprising two or preferably three domains or regions which are capable of binding to certain polysaccharides.
In one aspect the present disclosure relates to a polypeptide comprising at least two repeat regions selected from the group consisting of:
Y
V
12
13
W
E
F
35
36
38
Y
V
11
12
W
E
F
32
33
35
W
E
F
36
37
39
In one aspect the present disclosure relates to a polypeptide comprising at least two repeat regions selected from the group consisting of:
In one embodiment of the present disclosure, each repeat region of the polypeptide comprises a key Tryptophan (W) residue, preferably at position 35 of repeat region 1, position 32 of repeat region 2, and position 36 of repeat region 3, based on the amino acid numbering provided herein. Said Tryptophan (W) residue plays a key role in the binding between the repeat region that comprises it and a polysaccharide or disaccharide, as evident from the knock-out binding site mutants harbouring Tryptophan (W) to Alanine (A) substitution. The ring of the Tryptophan side chain has a major interaction with the carbon ring in a disaccharide or polysaccharide to which the polypeptide binds.
Thus, in one embodiment of the present disclosure, the polypeptide described herein comprises a repeat region 1, comprising an amino acid sequence SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions,
In another embodiment of the present disclosure, the polypeptide comprises a repeat region 2, comprising an amino acid sequence SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO: 65, SEQ ID NO: 68 or SEQ ID NO:221, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO: 65, SEQ ID NO: 68 or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions,
In another embodiment of the present disclosure, the polypeptide comprises a repeat region 3, comprising an amino acid sequence SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO: 51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66 or SEQ ID NO:69, SEQ ID NO:222, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO: 51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66 or SEQ ID NO:69, SEQ ID NO:222 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions,
In one embodiment of the present disclosure, each repeat region of the polypeptide comprises, in addition to a Tryptophan (W) residue at position 35 of R1, position 32 of R2 or position 36 of R3, also a Glutamic Acid at position 36 of R1, position 33 of R2 or position 37 of R3, and/or a Phenylalanine at position 38 of R1, position 35 of R2, or position 39 of R3, preferably both a Glutamic Acid at position 36 of R1, position 33 of R2 or position 37 of R3 and a Phenylalanine at position 38 of R1, position 35 of R2, or position 39 of R3, based on the amino acid numbering provided herein. Said Glutamic Acid and Phenylalanine residues are conserved and play an important role in the binding between the repeat regions that comprises them and a polysaccharide or disaccharide. This can also be seen in
Thus, in a further embodiment of the present disclosure, the polypeptide comprises a repeat region 1, comprising an amino acid sequence SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO: 40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO: 49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO: 64, SEQ ID NO:67 or SEQ ID NO:220, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO: 43, SEQ ID NO:46, SEQ ID NO: 49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO: 64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions,
In another embodiment of the present disclosure, the polypeptide comprises a repeat region 2, comprising an amino acid sequence SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO: 65, SEQ ID NO: 68 or SEQ ID NO:221, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO: 65, SEQ ID NO:68 or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions,
In another embodiment of the present disclosure, the polypeptide comprises a repeat region 3, comprising an amino acid sequence SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO: 51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO: 66, SEQ ID NO: 69 or SEQ ID NO:222, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO: 51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO: 66, SEQ ID NO: 69 or SEQ ID NO:222 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions,
In one embodiment of the present disclosure, each repeat region of the polypeptide comprises a Tyrosine at position 12 of R1 or at position 11 of R2 and R3 and/or a Valine at position 13 of R1 or at position 12 of R2 and R3 based on the amino acid numbering provided herein. These Tyrosine and/or Valine residues may contribute to binding of a repeat region to a disaccharide or polysaccharide, as they are located in proximity of the binding site in the 3D-structure of the polypeptide, when it binds to a disaccharide or polysaccharide.
In a further embodiment of the present disclosure, the polypeptide comprises a repeat region 1, comprising an amino acid sequence SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO: 64, SEQ ID NO:67 or SEQ ID NO:220, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 34, SEQ ID NO:37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO:46, SEQ ID NO: 49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO: 64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions,
In another embodiment of the present disclosure, the polypeptide comprises a repeat region 2, comprising an amino acid sequence SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO: 65, SEQ ID NO:68 or SEQ ID NO:221, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO: 65, SEQ ID NO: 68 or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions,
In another embodiment of the present disclosure, the polypeptide comprises a repeat region 2, comprising an amino acid sequence SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO: 51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO:222, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO: 51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO: 66, SEQ ID NO:69, SEQ ID NO:222 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions,
In one embodiment of the present disclosure, the repeat region 1 of the polypeptide disclosed herein comprises or consists of an amino acid sequence SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:220, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO: 64, SEQ ID NO:67, SEQ ID NO:220, in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions.
In one embodiment of the present disclosure, the repeat region 2 comprises or consists of an amino acid sequence SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO: 53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO: 68, SEQ ID NO:221, or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO: 53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO: 68, SEQ ID NO:221 in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions.
In one embodiment of the present disclosure, the repeat region 3 comprises or consists of an amino acid sequence SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO:51, SEQ ID NO: 54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO: 69, SEQ ID NO:222 or a functional variant thereof wherein the amino acid sequence of said variant differs from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO: 54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO: 69, SEQ ID NO:222, in that the amino acid sequence of the variant comprises 1 to 6 individual amino acid substitutions.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and repeat region 3 is flanked by at least three amino acid residues at its N-terminus and at least three amino acid residues at its C-terminus.
For example, in the polypeptide of the present disclosure each of repeat region 1, repeat region 2 and repeat region 3 is flanked by at least three amino acid residues at its N-terminus and at least three amino acid residues at its C-terminus so that:
YN—R1-YCZN—R2-ZCJN-R3-JC,
This structure is only one example and each of YN, YC, ZN, ZC, JN, and JC can individually be an amino acid sequence comprising more than 3 amino acids, such as more than, 5 amino acids, such as more than 10 amino acids, such as more than 15 amino acids, such as more than 20 amino acids. Furthermore, the number of amino acids in each of YN, YC, ZN, ZC, JN, and JC may be the same or different.
In one embodiment of the present disclosure, the functional variant SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO:67 or SEQ ID NO:220, in that the amino acid sequence of the variant comprises 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution.
In one embodiment of the present disclosure, the functional variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of the variant comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution at any one of residues 1 to 34 or 36 to 38 of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO: 43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO: 58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220.
In one embodiment of the present disclosure, the functional variant of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220 differs from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO:67 or SEQ ID NO:220 in that the amino acid sequence of the variant comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution at any one of residues 1 to 34 or 37 of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO: 58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 or SEQ ID NO:220.
In one embodiment of the present disclosure, the functional variant of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO: 62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221, differs from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO: 62, SEQ ID NO:65, SEQ ID NO: 68 or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution.
In one embodiment of the present disclosure, the functional variant of SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO: 62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 differs from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO: 62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution at any one of residues 1 to 31 or 33 to 35 of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO: 59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221.
In one embodiment of the present disclosure, the functional variant of SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:35, SEQ ID NO: 38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:68 or SEQ ID NO:221 differs from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:68 or SEQ ID NO:221 in that the amino acid sequence of the variant comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution at any one of residues 1 to 31 or 34 of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO: 44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO: 59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 or SEQ ID NO:221.
In one embodiment of the present disclosure, the functional variant of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO: 48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO: 63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 differs from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO: 48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO: 63, SEQ ID NO:66, SEQ ID NO: 69 or SEQ ID NO:222 in that the amino acid sequence of the variant comprises 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution.
In one embodiment of the present disclosure, the functional variant of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO: 48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO: 63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222 differs from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO: 48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69 or SEQ ID NO:222 in that the amino acid sequence of the variant comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution at any one of residues 1 to 35 or 37 to 39 of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO. 51, SEQ ID NO. 54, SEQ ID NO. 57, SEQ ID NO: 60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222.
In one embodiment of the present disclosure, the functional variant of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO: 48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO:69 or SEQ ID NO:222 differs from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO: 48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69 or SEQ ID NO:222 in that the amino acid sequence of the variant comprises 6 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 2 individual amino acid substitutions, such as 1 individual amino acid substitution at any one of residues 1 to 35 or 38 of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO: 45, SEQ ID NO:48, SEQ ID NO. 51, SEQ ID NO. 54, SEQ ID NO. 57, SEQ ID NO: 60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 or SEQ ID NO:222.
In one embodiment of the present disclosure, the individual amino acid substitutions are conservative amino acid substitutions.
In one embodiment of the present disclosure, the repeat region 1 of the polypeptide described herein comprises or consists of an amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO: 46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO: 61, SEQ ID NO:64, SEQ ID NO:67.
In one embodiment of the present disclosure, the repeat region 2 of the polypeptide described herein comprises or consists of an amino acid sequence of SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO: 62, SEQ ID NO:65, SEQ ID NO:68.
In one embodiment of the present disclosure, the repeat region 3 of the polypeptide described herein comprises or consists of an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO: 48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO: 63, SEQ ID NO:66, SEQ ID NO:69.
In one embodiment of the present disclosure, the polypeptide comprises repeat region 1 and repeat region 2, or functional variants thereof; repeat region 1 and repeat region 3, or functional variants thereof; repeat region 2 and repeat region 3, or functional variants thereof; repeat region 1, repeat region 2 and repeat region 3, or functional variants thereof.
In a further aspect the present disclosure relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20 to SEQ ID NO:33, or SEQ ID NO:71 to 219, and wherein said polypeptide is capable of binding one or more saccharide units.
In one embodiment of the present disclosure relates to a polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:20 to SEQ ID NO:33, and wherein said polypeptide is capable of binding one or more saccharide units.
In a further embodiment of the present disclosure, the polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 85% sequence identity to SEQ ID NO: 3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 90% sequence identity to SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 95% sequence identity to SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 20 to SEQ ID NO:33, such as at least 96% sequence identity to SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 97% sequence identity to SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33, such as at least 98% sequence identity to SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO: 33, such as at least 99% sequence identity to SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33.
In another embodiment of the present disclosure, the polypeptide comprises or consists of amino acid sequence SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:20 to SEQ ID NO:33.
In one embodiment of the present disclosure, the polypeptide comprises at least two repeat regions selected from the group consisting of:
In one embodiment of the present disclosure, the polypeptide comprises three repeat regions each selected from the group consisting of:
In an embodiment of the present disclosure, the polypeptide has a beta trefoil fold structure. Preferably, the structure consists of six β hairpins, each formed by two β strands, showing approximate three-fold symmetry.
A function of the polypeptide of the present disclosure is that it can bind certain disaccharides and polysaccharides and it can even cross-link certain polysaccharides. This function is connected at least partially to the presence in the polypeptides of the present disclosure of two or preferably three repeat regions, such as repeat region 1, repeat region 2 and/or repeat region 3, which are regions capable of recognizing and binding disaccharides and polysaccharides and are defined herein.
Thus, in one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and repeat region 3 is capable of binding at least one disaccharide.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and repeat region 3 is capable of binding at least one polysaccharide.
Despite the fact that they may have different amino acid sequences, the repeat regions of the polypeptides of the present disclosure are capable of binding disaccharides and polysaccharides characterized by comprising the same type of linkage between monosaccharide units.
The polypeptides of the present disclosure can bind any disaccharide or longer oligo- or polysaccharide as long as it comprises the specific Glc-β-1,6-linkage. A number of polysaccharides described herein comprise this linkage.
The polypeptides of the present disclosure also show binding to extremely rare Glc-β-1,2-Glc structures, which are found in unusual bacterial secretions.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding a disaccharide or polysaccharide comprising at least one Glc-β-1,6-Glc unit.
In another embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding a disaccharide or polysaccharide comprising at least one Glc-β-1,2-Glc unit.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding a disaccharide or polysaccharide comprising a glycan comprising at least one Glc-β-1,6-Glc unit.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding a disaccharide or polysaccharide having a β-1,6-glucan backbone.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding a disaccharide or polysaccharide having one or more Glc-β-1,6-Glc decoration.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding a disaccharide or polysaccharide having one or more Glc-β-1,6-Glc decoration(s) on a β-1,3-glucan backbone.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding the polysaccharide scleroglucan.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding the polysaccharide pustulan.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding the polysaccharide laminarin.
In one embodiment of the present disclosure, each of repeat region 1, repeat region 2 and/or repeat region 3 is capable of binding the polysaccharide gentiobiose.
In one embodiment of the present disclosure, the saccharide is at least a disaccharide.
In one embodiment of the present disclosure, the saccharide is an oligosaccharide or a polysaccharide.
In one aspect the present disclosure relates to a polynucleotide encoding a polypeptide described herein.
In one embodiment of the present disclosure the polynucleotide encodes a polypeptide comprising at least two repeat regions selected from the group consisting of:
In one embodiment of the present disclosure the polynucleotide is codon-optimized for expression in a host cell.
In another aspect the present disclosure relates to a recombinant vector comprising a polynucleotide described herein, or a nucleic acid sequence encoding a carbohydrate-binding polypeptide according to any of the embodiments described herein. Examples of a recombinant vector include but not limited to plasmids, viral vectors, cosmids, and artificial chromosomes.
In one embodiment of the present disclosure the vector is a plasmid vector.
In a further aspect the present disclosure relates to a host cell (e.g. standard expression strains of E. coli) expressing the recombinant vector as described herein.
As provided herein, the polypeptides of the present disclosure are capable of binding polysaccharides. Each polypeptide of the present disclosure can bind up to three chains of polysaccharides. Moreover, if several polypeptides of the present disclosure are present together with the polysaccharides, the polypeptides of the present disclosure may be capable of not only binding said polysaccharides but also cross-linking them. The polypeptides of the present disclosure are thus capable of binding and cross-linking certain polysaccharides; the specific polysaccharides are described herein.
In one aspect the present disclosure relates to use of the polypeptide described in any of the embodiments herein for cross-linking two or more polysaccharides.
Another aspect of the present disclosure relates to a composition comprising at least one polypeptide according to any of the embodiments described herein and two or more polysaccharides, wherein the polysaccharides are cross-linked by the polypeptide, such as wherein the polypeptide is the cross-linker linking the polysaccharides together.
In another aspect the present disclosure relates to a hydrogel comprising at least one polypeptide according to any of the embodiments described herein and two or more polysaccharides, wherein said hydrogel has a cross-linked structure.
In another aspect the present disclosure relates to a hydrogel comprising at least two polypeptides according to any of the embodiments described herein and three or more polysaccharides, wherein said hydrogel has a cross-linked structure.
Preferably, the present disclosure also relates to a hydrogel comprising a plurality of polypeptides according to any of the embodiments described herein and a plurality of polysaccharides, wherein said hydrogel has a cross-linked structure.
In fact, the polypeptides according to any of the embodiments described herein function as cross-linkers in the hydrogel of the present disclosure.
In one embodiment of the present disclosure, the hydrogel is cross-linked with a bi- or multifunctional linker, for example a linker capable of binding to two or more polysaccharide chains, which is a polypeptide as described herein.
In one embodiment of the present disclosure, the hydrogel comprises one or more polysaccharides each having a size of 20.000 Da or more, such as of 50.000 Da or more, such as of 70.000 Da or more, such as of 100.000 Da or more, such as of 150.000 Da or more, such as of 200.000 Da or more, such as of 300.000 Da or more, such as of 500.000 Da or more, such as of 700.000 Da or more, such as of 800.000 Da or more, such as of 900.000 Da or more, such as of 1.000.000 Da or more, such as where the polysaccharide has a size of about 2.000.000 Da Preferably, the polysaccharide at least 100.000 Da, such as at least 500.000 Da, such as at least 1.000.000 Da, such as 2.000.000 Da.
In one embodiment of the present disclosure, the hydrogel comprises a polysaccharide that is water-dispersible. In a further embodiment, the polysaccharide has a moisture content of at least 70 μg water per mg of saccharide, preferably at least 74 μg water per mg of saccharide, such as 77 μg water per mg of saccharide.
In one embodiment of the present disclosure, the hydrogel comprises one or more polysaccharides, which are biopolymers, each comprising one or more β-1,3-glucan units with β-1,6-linked glucose decorations to the main chain, such as scleroglucan or schizophyllan.
In one embodiment of the present disclosure, the hydrogel comprises between 0.1 g/L and 10 g/L of a polypeptide of the present disclosure, and between 2 g/L and 10 g/L of a polysaccharide defined herein. Preferably, the hydrogel comprises between 0.15 g/L and 5 g/L of a polypeptide of the present disclosure, and between 3 g/L and 5 g/L of a polysaccharide defined herein.
In one embodiment of the present disclosure, the hydrogel comprises at least 0.1 g/L of the polypeptide of the present disclosure, such as at least 0.2 g/L polypeptide, such as at least 0.3 g/L polypeptide, such as at least 0.5 g/L polypeptide, such as at least 0.8 g/L polypeptide, such as at least 1 g/L polypeptide, such as at least 1.3 g/L polypeptide, such as at least 1.7 g/L polypeptide, such as at least 2 g/L polypeptide, such as at least 2.5 g/L polypeptide, such as at least 3 g/L polypeptide, such as at least 3.5 g/L polypeptide, such as at least 4 g/L polypeptide, such as at least 4.5 g/L polypeptide, such as at least 5 g/L polypeptide, such as at least 5.5 g/L polypeptide, such as at least 6 g/L polypeptide, such as at least 6.5 g/L polypeptide, such as at least 7 g/L polypeptide, such as at least 7.5 g/L polypeptide, such as at least 8 g/L polypeptide, such as at 8.5 g/L polypeptide, such as at least 9 g/L polypeptide, such as at least 9.5 g/L polypeptide, such as at least 10 g/L of a polypeptide of the present disclosure.
In one embodiment of the present disclosure, the hydrogel comprises at the most 10 g/L of a polypeptide of the present disclosure.
In one embodiment of the present disclosure, the hydrogel comprises at least 2 g/L polysaccharide, such as at least 3 g/L polysaccharide, and at the most 5 g/L polysaccharide, such as at the most 6 g/L polysaccharide, such as at the most 7 g/L polysaccharide, such as at the most 8 g/L polysaccharide, such as at the most 9 g/L polysaccharide, 10 g/L polysaccharide. Preferably, in an embodiment of the present disclosure, the hydrogel comprises at least 3 g/L polysaccharide, and at the most 5 g/L polysaccharide, wherein the polysaccharide comprises at least one Glc-β-1,6-Glc unit.
In one embodiment of the present disclosure, the hydrogel comprises at least 2.5 g/L polysaccharide, such as at least 3 g/L polysaccharide, such as at least 3.5 g/L polysaccharide, such as at least 4 g/L polysaccharide, such as at least 4.5 g/L polysaccharide, such as at least 5 g/L polysaccharide, such as at least 5.5 g/L polysaccharide, such as at least 6 g/L polysaccharide, such as at least 6.5 g/L polysaccharide, such as at least 7 g/L polysaccharide, such as at least 7.5 g/L polysaccharide, such as at least 8 g/L polysaccharide, such as at 8.5 g/L polysaccharide, such as at least 9 g/L polysaccharide, such as at least 9.5 g/L polysaccharide, such as at the most 10 g/L of a polysaccharide, wherein the polysaccharide comprises at least one Glc-β-1,6-Glc unit.
In one embodiment of the present disclosure, the hydrogel comprises at least 2 g/L Scleroglucan, such as at least 3 g/L Scleroglucan, and at the most 10 g/L
Scleroglucan, such as at the most 6 g/L Scleroglucan, such as at the most 7 g/L Scleroglucan, such as at the most 8 g/L Scleroglucan, such as at the most 9 g/L Scleroglucan. Preferably, in an embodiment of the present disclosure, the hydrogel comprises at least 3 g/L Scleroglucan, and at the most 5 g/L Scleroglucan.
In one embodiment of the present disclosure, the hydrogel comprises at least 2.5 g/L Scleroglucan, such as at least 3 g/L Scleroglucan, such as at least 3.5 g/L Scleroglucan, such as at least 4 g/L Scleroglucan, such as at least 4.5 g/L Scleroglucan, such as at least 5 g/L Scleroglucan, such as at least 5.5 g/L Scleroglucan, such as at least 6 g/L Scleroglucan, such as at least 6.5 g/L Scleroglucan, such as at least 7 g/L Scleroglucan, such as at least 7.5 g/L Scleroglucan, such as at least 8 g/L Scleroglucan, such as at 8.5 g/L Scleroglucan, such as at least 9 g/L polysaccharide, such as at least 9.5 g/L polysaccharide, such as at the most 10 g/L of a polysaccharide, wherein the polysaccharide comprises at least one Glc-β-1,6-Glc unit.
For example, as illustrated in
In one embodiment of the present disclosure, the hydrogel comprises at least 90 wt. % water, such as at least 91 wt. % water, such as at least 92 wt. % water, such as at least 93 wt. % water, such as at least 94 wt. % water, such as at least 95 wt. % water, such as at least 96 wt. % water, such as at least 97 wt. % water, such as at least 98 wt. % water, such as at least 99 wt. % water, such as at least 99.5 wt. % water.
The hydrogel according to one embodiment of the present disclosure is able to dry out in air at room temperature and subsequently of being rehydrated, if put in contact with water. In one embodiment, the rehydrated gel has a lower water content that the original hydrogel, for example, the rehydrated gel may comprise 70 wt. % to 90 wt. % water. For example, the rehydrated hydrogel may be in the form of a film.
In one embodiment, the rehydrated gel has a water content that resembles that of the original hydrogel, for example, the rehydrated gel may comprise at least 70 wt. % water, such as at least 80 wt. % water, such as at least 90 wt. % water.
In an embodiment of the present disclosure the hydrogel can be dehydrated, retaining cross-linked structure, and re-hydrated or re-swelled again. By the processes of dehydration and rehydration is meant the process of alteration of the physical form of the hydrogel due to loss of water and restoration of water content, such as re-swelling, respectively.
Dehydration and rehydration of the hydrogel of the present disclosure may be conducted by any of the methods known in the art.
In an embodiment of the present disclosure, the hydrogel is a re-hydrated hydrogel and the re-hydrated hydrogel comprises at least 70 wt. % water, such as at least 73 wt. % water, such as at least 75 wt. % water, such as at least 78 wt. % water, such as at least 80 wt. % water, such as at least 83 wt. % water, such as at least 85 wt. % water, such as at least 88 wt. % water, such as at least 90 wt. % water.
In an embodiment of the present disclosure, the hydrogel comprises the polypeptide of the present disclosure and a polysaccharide at a ratio of polypeptide to polysaccharide between 1:20 to 1:2, such as at a ratio between 1:20 and 1:3, such as at a ratio between 1:20 and 1:5, such as at a ratio between 1:20 and 1:7, such as at a ratio between 1:20 and 1:10, such as at a ratio between 1:20 and 1:12, such as at a ratio between 1:20 and 1:15, such as at a ratio between 1:20 and 1:18, such as at a ratio between 1:18 and 1:2, such as at a ratio between 1:15 and 1:2, such as at a ratio between 1:13 and 1:2, such as at a ratio between 1:10 and 1:2, such as at a ratio between 1:8 and 1:2, such as at a ratio between 1:6 and 1:2, such as at a ratio between 1:4 and 1:2. For example, the polysaccharide may be scleroglucan. These ratios are volume:volume ratios based on a stock solution of polysaccharide of 5 g/L in water at room temperature, and a stock solution of a polypeptide of the present disclosure of 4 g/l in water at room temperature.
In an embodiment of the present disclosure, the hydrogel comprises:
In an embodiment of the present disclosure, the hydrogel comprises:
Y
V
12
13
W
E
F
35
36
38
Y
V
11
12
W
E
F
32
33
35
W
E
F
36
37
39
In an embodiment of the present disclosure, the hydrogel comprises:
In an embodiment of the present disclosure, the hydrogel comprises:
Y
V
12
13
W
E
F
35
36
38
Y
V
11
12
W
E
F
32
33
35
W
E
F
36
37
39
In an embodiment of the present disclosure, the hydrogel comprises:
In an embodiment of the present disclosure, the hydrogel comprises:
In an embodiment of the present disclosure, the hydrogel comprises:
In an embodiment of the present disclosure, the hydrogel comprises:
In another aspect the present disclosure relates to a cross-linker for crosslinking polysaccharides to form hydrogels, wherein said cross-linker is a polypeptide as described herein.
In yet another aspect the present disclosure relates to a method of manufacturing a hydrogel, said method comprising:
In an embodiment of the present disclosure, the hydrogel comprises a polysaccharide as described herein.
Database searches for CBM domain homologues were carried out using BLASTP with C. pinensis Cp-F1 protein as query against the non-redundant protein sequences dataset of the Genbank database at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). Sequences containing homologues to Cp-F1 were selected to generate a CBM containing-protein subset for further analysis. This subset was evaluated using the taxonomy browser at NCBI (http://www.ncbi.nlm.nih.gov/Taxonomy). Incomplete and redundant entries were removed. Additionally, only one exemplary species was selected from each genus, and the final dataset contained 164 sequences.
All identified carbohydrate binding protein modules are summarized in Table 1. The table contains gene accession numbers, species name, abbreviated identifier, and sequence identity number refereeing to the ST.26 sequence listing enclosed, for all carbohydrate binding protein modules identified. The identifiers comprise an abbreviation of the species name and the number of amino acids in the full-length multi-modular protein that contains one of the carbohydrate binding domains. In native form, these carbohydrate binding domains are all part of multi-modular proteins with carbohydrate-degrading functions.
Aquimarina aggregata
Aquimarina aggregata
Aquimarina aggregata
Aquimarina aggregata
Aquimarina aggregata
Aquimarina aggregata
Algibacter aquaticus
Algibacter aquaticus
Armatimonadetes bacterium
Actinobacteria bacterium
Acidimicrobiia bacterium
Actinobacteria bacterium
Actinoplanes globisporus
Apiotrichum porosum
Actinospica robiniae
Anaeromicrobium sediminis
Blastocatellia bacterium
Blastocatellia bacterium
Blastocatellia bacterium
Blastocatellia bacterium
Burkholderia gladioli
Bacillus solimangrovi
Coraliomargarita akajimensis DSM
Coraliomargarita akajimensis DSM
Coraliomargarita akajimensis DSM
Coraliomargarita akajimensis DSM
Capsulimonas corticalis
Coleofasciculus sp. FACHB-129
Chryseolinea flava
Chryseolinea flava
Chryseolinea flava
Chryseolinea flava
Chryseolinea flava
Coptotermes formosanus
Cystobacter fuscus
Comamonas sp. KCTC 72670
Candidatus Poseidoniales
archaeon
Candidatus Poseidoniales
archaeon
Candidatus Poseidoniales
archaeon
Candidatus Poseidoniales
archaeon
Chitinophaga pinensis DSM 2588
Chitinophaga pinensis DSM 2588
Chitinophaga pinensis DSM 2588
Chitinophaga pinensis DSM 2588
Chitinophaga pinensis DSM 2588
Chitinophaga pinensis DSM 2588
Chitinophaga pinensis DSM 2588
Catenulispora rubra
Chengkuizengella sediminis
Camellia sinensis
Cylindrospermum stagnale
Corallococcus terminator
Cellulomonas sp. URHD0024
Dictyobacter alpinus
Dictyobacter kobayashii
Draconibacterium sp. GM2-18
Dinghuibacter silviterrae
Deminuibacter soli
Dactylosporangium sucinum
Dichotomicrobium
thermohalophilum
Dictyobacter vulcani
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Euryarchaeota archaeon
Fibrella aestuarina
Flavobacteriaceae bacterium M625
Flavobacteriaceae bacterium M625
Fischerella muscicola
Flavobacterium subsaxonicum
Geomonas soli
Herpetosiphon aurantiacus DSM
Hyphomicrobiaceae bacterium
Hymenobacter sp. BRD128
Herpetosiphon llansteffanensis
Hyalangium minutum
Haliangium ochraceum
Hamadaea tsunoensis
Hydra vulgaris
Kordia jejudonensis
Kordia jejudonensis
Ktedonobacter racemifer
Microcystis aeruginosa
Marinoscillum sp. 108
Myxococcaceae bacterium
Mangrovibacterium diazotrophicum
Myxococcus hansupus
Marivirga lumbricoides
Marivirga lumbricoides
Mitsuaria noduli
Micromonospora orduensis
Micromonospora orduensis
Micromonospora orduensis
Micromonospora orduensis
Mucilaginibacter rubeus
Mucilaginibacter rubeus
Mucilaginibacter rubeus
Mucilaginibacter rubeus
Mucilaginibacter rubeus
Nonomuraea angiospora
Niastella koreensis
Niastella koreensis
Niastella koreensis
Niastella koreensis
Nakamurella multipartita
Nostoc parmelioides
Nonlabens xiamenensis
Ohtaekwangia koreensis
Ohtaekwangia koreensis
Ohtaekwangia koreensis
Phycisphaerales bacterium
Phycisphaerales bacterium
Phycisphaerae bacterium
Plantactinospora sp. BB1
Pyxidicoccus sp. CA060A
Pyxidicoccus sp. CA060A
Pedobacter duraquae
Pedobacter duraquae
Phytohabitans flavus
Pseudarcicella hirudinis
Paenibacillus hunanensis
Paucimonas lemoignei
Plantactinospora soyae
Paraflavitalea soli
Pseudoflavitalea sp. X16
Rickettsiales bacterium
Rickettsiales bacterium
Rickettsiales bacterium
Roseateles depolymerans
Roseateles depolymerans
Rhizobacter sp. OV335
Rhododendron simsii
Reichenbachiella versicolor
Stigmatella aurantiaca
Saccharicrinis aurantiacus
Saccharicrinis aurantiacus
Saccharicrinis aurantiacus
Saccharicrinis aurantiacus
Syntrophaceae bacterium
Saprospirales bacterium
Saccharicrinis carchari
Sediminitomix flava
Sesamum indicum
Sinomicrobium pectinilyticum
Sunxiuqinia sp. RC1_OXG_1F
Symploca sp. SIO2E6
Senna tora
Trinickia sp. 7GSK02
Tenacibaculum sp. M341
Tenacibaculum sp. M341
Terriglobus saanensis
Verrucomicrobia bacterium
Verrucomicrobia bacterium
Verrucomicrobia bacterium
Vitiosangium sp. GDMCC 1.1324
Zobellia sp. O113
Certain genes explored in this study were synthesised in a proprietary vector by ThermoFisher GeneArt; these were then sub-cloned into the expression vector pET21a (ThermoFisher), which carries a C-terminal His6-tag and confers ampicillin resistance (marked as “Commercial synthesis” in Table 2). Other genes were cloned in-house from genomic C. pinensis DNA (DSMZ, Germany) (marked as “Cloned from gDNA into PLATE31” in Table 2).
Recombinant proteins were generated according to the cloning strategy described in Table 2. CBM domains were cloned from large multi-modular genes. The range of nucleotides specified therefore refers to the fragment that was cloned from the full-length gene. The size (kDa) refers to the resulting recombinant CBM protein. gDNA=genomic DNA.
Chitinophaga
pinensis
Chitinophaga
pinensis
Chitinophaga
pinensis
Chitinophaga
pinensis
Chitinophaga
pinensis
Chitinophaga
pinensis
Chitinophaga
pinensis
Chitinophaga
pinensis
Draconibacterium
mangrovi
Aquimarina
aggregata
Euryarchaeota
archaeon
Aquimarina
aggregata
Pyxidicoccus
caerfyrddinensis
Results: Protein production and purification was analysed by SDS-PAGE every time. Protein yield measured every time by Bradford assay and/or by using a nanodrop. SDS-PAGE analysis confirmed successful production and purification for all constructs (data not shown). Typical protein yield for Cp-F1 is 150 mg pure protein from a 1 L culture of over-expressing E. coli cells. Other proteins were produced and purified with a yield of 25-75 mg and up to 150 mg pure protein from a 1 L culture.
Binding to water-insoluble polysaccharides for 12 proteins identified as part of the CBM92 family (see Table 3) as well as five F1 variants where the Tryptophan (W/Trp) in the binding site “WExF” motif was converted by site-directed mutagenesis to an Alanine (A/Ala), in either one (α, β or γ), two (β/γ) or three (α/β/γ) binding domains was tested by pull-down assay (
Results: The absence of a protein band in SDS-PAGE indicated that the protein successfully bound to the polysaccharide (Table 4). A consistent affinity for binding to polysaccharides containing the Glc-β-1,6-Glc linkage, namely pustulan (linear β-1,6-glucan), as well as scleroglucan and yeast β-glucan (both consisting of β-1,3-glucan chains with single substitutions of β-1,6-linked glucosyl residues) was shown. Some of the CBM proteins tested also showed some binding to curdlan, a linear β-1,3-glucan, and in some cases there was binding to lichenan, which comprises β-1,3- and β-1,4-linked glucosyl residues. There was also weak binding to birchwood xylan in some cases, suggesting the capacity for more relaxed specificity in certain CBM proteins. Of note, Dm-F1, which naturally lacks all binding-site Trp residues, did not bind any of the polysaccharides tested (
All wild type proteins that showed binding with scleroglucan were also confirmed to be gel-forming.
Sequence analysis of Cp-F1 (SEQ ID NO:3, SEQ ID NO:20) and Cp-F2 (SEQ ID NO:4, SEQ ID NO:21) as well as its homologues) showed three repeat regions, SEQ ID NOs: 8, 10 and 12 for Cp-F1, and SEQ ID Nos: 9, 11 and 13 for Cp-F2, each containing one conserved Tryptophan binding site. The same repeat regions were confirmed in the other proteins of the CBM92 family (the proteins corresponding to SEQ ID NO:22 to SEQ ID NO:33 of Table 1), each having three repeat regions corresponding to the amino acid sequences of SEQ ID NO:34 to SEQ ID NO:69. Structural analysis of the protein by X-ray crystallography showed three apparent binding pockets, two of which have been able to be crystallised with a Glucose monosaccharide ligand. All three putative binding sites have been explored and confirmed by site-directed mutagenesis. Rheological analysis of mutant variants of the Cp-F1 protein showed that all three binding sites must be functional for stable hydrogel formation to occur. For each mutation, the Tryptophan (W/Trp) in the binding site “WExF” motif was converted by site-directed mutagenesis to an Alanine (A/Ala), the experiments were performed with a final in-gel scleroglucan concentration of 4 g/L and 50% protein loading (i.e. final protein concentration of 2 g/L).
Protein Cp-F1 at a starting stock concentration of 4 mg/ml (higher starting stock concentrations can also be used) in water was mixed with scleroglucan at a starting stock concentration of 5 g/L in water at room temperature. The two solutions were mixed together quickly by adding the protein solution into a continuously vortexed polysaccharide solution. A hydrogel formed spontaneously within a few seconds and reached maximum stiffness after around one hour. In order to alter the stiffness of the hydrogel, we used a consistent stock concentration for both scleroglucan (e.g., 5 mg/ml as stock solution) and protein (4-30 mg/ml as stock solution), and mixed these with different ratios. The following final protein concentrations were tested: 0.08 g/L, 0.2 g/L, 0.4 g/L, 0.8 g/L, 1.2 g/L, 1.6 g/L and 2 g/L, which is equivalent to 2%, 5%, 10%, 20%, 30%, 40% and 50% of the protein content relative to the polysaccharide content respectively (see
Another way would be to use a consistent scleroglucan concentration (e.g., 4 mg/ml in the final mixture, 5 mg/ml in the starting stock solution), and vary protein concentration by preparing differently concentrated protein stock solutions in advance.
Example: to produce 1 ml of hydrogel with final concentrations of 4 mg/ml scleroglucan and 2 mg/ml F1 protein, we mixed 0.8 ml scleroglucan (a solution at 5 mg/ml in water) with 0.2 ml F1 protein (a solution of 10 mg/ml in water).
Rheological properties of the hydrogel were measured at 25° C. using a rheometer with cone-plate configuration. Multiple concentrations and ratios of protein and polysaccharide were measured for each gel, always comparing to a rheology measurement for “polysaccharide without protein”. Both the shear viscosity and the dynamic shear properties, given by the storage modulus G′ and the loss modulus G″, were determined for each rheological experiment.
Results: When Cp-F1 or Cp-F2 were mixed with scleroglucan, a hydrogel formed spontaneously within a few minutes and almost reached maximum stiffness around one hour (
When increasing the protein:saccharide ratio, solidity of the gel increased and transparency decreased (
| Number | Date | Country | Kind |
|---|---|---|---|
| 21208133.5 | Nov 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/081942 | 11/15/2022 | WO |
