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WO2019046943A1 - Protéines de type collagène - Google Patents

Protéines de type collagène Download PDF

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Publication number
WO2019046943A1
WO2019046943A1 PCT/CA2018/051077 CA2018051077W WO2019046943A1 WO 2019046943 A1 WO2019046943 A1 WO 2019046943A1 CA 2018051077 W CA2018051077 W CA 2018051077W WO 2019046943 A1 WO2019046943 A1 WO 2019046943A1
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Prior art keywords
polypeptide
collagen
domain
protein
hycoll
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English (en)
Inventor
James Leroy HARDEN
Kim MERRETT
Chyan-Jang LEE
Fan WAN
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University of Ottawa
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University of Ottawa
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A61F2/105—Skin implants, e.g. artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/14—Eye parts, e.g. lenses or corneal implants; Artificial eyes
    • A61F2/142—Cornea, e.g. artificial corneae, keratoprostheses or corneal implants for repair of defective corneal tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/416—Anti-neoplastic or anti-proliferative or anti-restenosis or anti-angiogenic agents, e.g. paclitaxel, sirolimus

Definitions

  • the present invention relates generally to collagen. More specifically, the present invention relates to collagen-like proteins and portions thereof, as well as related
  • compositions for purposes of this specification.
  • Collagen is the most abundant protein in the extracellular matrix (ECM) of mammals that surrounds cells and forms the cell-interactive scaffolding of the body.
  • ECM extracellular matrix
  • the defining feature of collagen is its molecular structure, including a unique super coiled triple-helix.
  • Streptococcus pyogenes is known to produce a cell surface collagen-like protein (Scl2) that contains an N-terminal globular V domain, followed by a collagen-like CL domain and a C- terminal trans-membrane protein (Xu, Y., Keene, D.R., Bujnicki, J.M., Hook, M. and
  • the N-terminal globular V domain is an a-helix containing protein that forms trimers and has been shown to facilitate triple-helix folding.
  • the collagenous domain (CL) is an acidic highly charged protein consisting of the repeated amino acid sequence Gly-Xaa-Yaa in which the glycine residue occupies every third position, since only glycine is small enough to be accommodated at the center of the triple helix.
  • polypeptide comprising a sequence with at least 70% sequence identity to the sequence:
  • the polypeptide comprises a sequence with at least 75%, 80%, 85%,
  • polypeptide comprises the sequence of SEQ ID NO: 1 or a fragment thereof.
  • polypeptide comprises the sequence of SEQ ID NO: 1.
  • the polypeptide further comprises a bioactive domain.
  • the bioactive domain comprises at least one of a cell binding domain, a cell signalling domain, a biomineralization domain, a metallization domain, an elastomeric domain, and a biomimetic structural protein.
  • the cell binding domain comprises at least one of RGD, RGDS, DGEA, REDV, YIGSR, IKVAV, KAFAK, and VAPG.
  • the biomineralization domain comprises at least one of MLPHHGA, SVSVGMKPSPRP, ESQES, and QESQSEQDS.
  • the metallization domain comprises at least one of NPSSLFRYLPSD, MHGKTQATSGTIQS, AQNPSDNNTHTH, and RLELAIPLQGSG.
  • the elastomeric domain comprises at least one of VPGVG, (AG) 3 EG,
  • GYSGGRPGGQDLG GGFGGMGGGS, PGQGQQ, and GYYPTSPQ.
  • bioactive domain is linked to the C-terminal end of the polypeptide.
  • bioactive domain is linked to the polypeptide with a linker sequence.
  • the linker sequence comprises (GSTSGSGT) n or
  • polypeptide is modified to include cross-linking elements.
  • the cross-linking elements are primary amines, aromatics, and/or glutamine.
  • the cross-linking elements are selected from lysine, tyrosine, tryptophan, phenylalanine, glutamine, histidine and combinations thereof.
  • the polypeptide further comprises one or more terminal cysteines for conjugation to other polymers and/or peptides.
  • the polypeptide is ionically crosslinked.
  • the polypeptide is ionically crosslinked with cationic and/or anionic crosslinkers, such as tri-poly-phosphate or spermidine.
  • polypeptide comprising the sequence
  • each X is independently selected from lysine, tyrosine, tryptophan, phenylalanine, histidine and glutamine.
  • polypeptide comprising the sequence
  • GPXGEQGPQGLPGKDGEAGAQGPAGPMGPAGEXGEKGEPGYXGAKGDRGETGPXGPX GERGEAGPAGKDGERGPVGPA or a sequence having at least 70% sequence identity thereto, wherein each X is independently selected from lysine, tyrosine, tryptophan, phenylalanine, histidine and glutamine.
  • polypeptide comprising the sequence
  • GPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPR GERGEAGPAGKDGERGPVGPA or a sequence having at least 70% sequence identity thereto, wherein at least one amino acid residue is substituted with a lysine, a tyrosine, a tryptophan, a phenylalanine, a histidine, or a glutamine.
  • polypeptide is repeated consecutively 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.
  • the polypeptide further comprises a V domain of a collagen-like protein, wherein the V domain is optionally cleaved after expression of the polypeptide.
  • a polypeptide encoding a modified collagen-like protein or fragment thereof, wherein the modified collagen-like protein is modified to increase availability of primary amines, aromatics, and/or glutamine for crosslinking.
  • the modified collagen-like protein is modified to increase availability of lysine, tyrosine, tryptophan, phenylalanine, glutamine, histidine or combinations thereof.
  • a collagen-like protein comprising the polypeptide described herein.
  • the collagen-like protein is of bacterial origin and is modified to comprise the polypeptide described herein.
  • the collagen-like protein is derived from Streptococcus pyogenes. In an aspect, the collagen-like protein is a modified Scl2 protein.
  • a naturally occurring portion of the collagen-like protein is substituted with the polypeptide described herein.
  • the collagen-like protein is a V-CL protein comprising an N-terminal globular V domain, followed by a collagen-like CL domain, wherein the CL domain comprises at least one copy of the polypeptide described herein.
  • the CL domain consists of at least one copy of the polypeptide described herein.
  • the polypeptide described herein is present in one or more substantially consecutive repeats.
  • a collagen-like CL domain of a collagen-like protein comprising the polypeptide described herein.
  • the CL domain is of bacterial origin and is modified to comprise the polypeptide described herein.
  • the CL domain is derived from Streptococcus pyogenes.
  • the CL domain is a modified Scl2 CL domain.
  • a naturally occurring portion of the CL domain is substituted with the polypeptide described herein.
  • the CL domain is fused to an N-terminal globular V domain, wherein the CL domain comprises at least one copy of the polypeptide described herein.
  • the CL domain consists of at least one copy of the polypeptide described herein.
  • polypeptide described herein is present in one or more substantially consecutive repeats.
  • nucleic acid molecule encoding the polypeptide, the collagen-like protein, or the CL domain described herein.
  • an expression vector comprising the nucleic acid molecule described herein.
  • a recombinant host cell comprising the expression vector described herein.
  • a recombinant host cell expressing, displaying, and/or secreting the polypeptide, the collagen-like protein, or the CL domain described herein.
  • composition comprising the polypeptide, the collagen-like protein, or the CL domain described herein.
  • composition further comprises a pharmaceutically acceptable carrier, diluent, and/or buffer.
  • the composition is transparent.
  • the composition is a hydrogel.
  • the hydrogel is produced by chemical, physical, ionic, photo, or enzyme based crosslinking methods.
  • the hydrogel is thermally stable at physiologic temperature (37°C).
  • the composition further comprises at least one of cells, growth factors, nanoparticles, other proteins and polymers.
  • a bio-ink comprising the composition described herein.
  • an ophthalmic device comprising the composition described herein.
  • a dermal implant comprising the composition described herein.
  • Figure 1 The nucleic acid and polypeptide sequence of the HyColl Sequence.
  • FIG. 1 Cell binding designs: (A) HyColl-RGD Sequence; (B) HyColl-S-RGD with a GSTSGSGT (spacer, S).
  • FIG. 3A A generic biofunctional HyColl design with an optional linker/spacer sequence YYYY of variable length, an optional bioactive domain XXXXi, an optional mineralization binding domain XXXX 2 , an optional metal binding domain XXXX 3 , and/or an optional elastomeric domain ZZZZ.
  • FIG. 3B A generic biofunctional HyColl design showing modifications to include crosslinking elements.
  • FIG. 4 A Gelcode blue stained SDS-PAGE gel showing the purification of HyColl and HyColl-RGD.
  • the lanes contain the following: Lane 1 : Precision plus proteinTM standards; Lane 2: Purified HyColl; Lane 3: Purified HyColl-RGD.
  • CD wavelength scans at 10 ⁇ HyColl-RGD solutions showing an ellipticity maximum at 220nm and a minimum at 198nm characteristic of triple helix secondary structure in acidic, neutral, and basic pH buffers.
  • FIG. 9 CD thermal scans of HyColl in citric acid and Na2HPC (pH 4) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).
  • Figure 10 CD Thermal scans of HyColl in citric acid and Na 2 HP0 4 (pH 7) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).
  • Figure 1 CD thermal scans of 10 ⁇ HyColl in NaH 2 P0 4 and Na 2 HP0 4 (pH 7) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).
  • FIG. 14 CD thermal scans of 10 ⁇ HyColl-RGD in citric acid and Na 2 HP0 4 (pH 4) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).
  • Figure 17 CD thermal scans of 10 ⁇ HyColl-RGD in PBS buffer (pH 7.4) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).
  • FIG. 1 CD thermal scans of 10 ⁇ HyColl-RGD in NaHC0 3 and Na 2 C0 3 (pH 10) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).
  • FIG. 19 CD wavelength scans of 10 ⁇ HyColl in citric acid and Na 2 HP0 (pH 7) at 20°C, 30°C, 40°C and 50°C showing that the protein is denatured at 40°C. After 24 hour recovery, the CD signal reached 99% of the original signal demonstrating refolding.
  • FIG. 20 Micro-DSC heating and cooling scan for 100 ⁇ HyColl in Na 2 HP0 and citric acid (pH 4).
  • FIG 21 Micro-DSC heating and cooling scan for 100 ⁇ HyColl in MES (pH 4.5).
  • Figure 22 Micro-DSC heating and cooling scan for 100 ⁇ HyColl in Na 2 HP0 and citric acid (pH 7).
  • Figure 23 Micro-DSC heating and cooling scan for 100 ⁇ HyColl in Na 2 HP0 4 and NaH 2 P0 4 (pH 7).
  • FIG. 24 Micro-DSC heating and cooling scan for 100 ⁇ HyColl in Na 2 C0 3 and NaHC0 3 (pH 10).
  • FIG. 25 Micro-DSC heating and cooling scan for 10 ⁇ HyColl in Na 2 HP0 4 and citric acid (pH 4).
  • FIG 26 Micro-DSC heating and cooling scan for 10 ⁇ HyColl in MES (pH 4.5).
  • Figure 27 Micro-DSC heating and cooling scan for 10 ⁇ HyColl in Na 2 HP0 4 and citric acid (pH 7).
  • FIG. 29 Micro-DSC heating and cooling scan for 10 ⁇ HyColl in Na 2 C0 3 and NaHC0 3 (pH 10).
  • FIG. 30 Micro-DSC heating and cooling scan for 100 ⁇ HyColl-RGD in Na 2 HP0 4 and citric acid (pH 4).
  • FIG 31 Micro-DSC heating and cooling scan for 100 ⁇ HyColl-RGD in Na 2 HP0 and citric acid (pH 7).
  • FIG. 33 Micro-DSC heating and cooling scan for 100 ⁇ HyColl-RGD Na 2 HP0 4 and NaH 2 P0 4 (pH 7).
  • FIG. 34 Micro-DSC heating and cooling scan for 100 ⁇ HyColl-RGD in Na 2 C03 and NaHC0 3 (pH 10).
  • FIG. 35 Micro-DSC heating and cooling scan for 10 ⁇ HyColl-RGD in Na 2 HP0 and citric acid (pH 4).
  • FIG. 36 Micro-DSC heating and cooling scan for 10 ⁇ HyColl-RGD in Na 2 HP0 and citric acid (pH 7).
  • FIG 38 Micro-DSC heating and cooling scan for 10 ⁇ HyColl-RGD in Na 2 HP0 and NaH 2 P0 4 (pH 7).
  • FIG 39 Micro-DSC heating and cooling scan for 10 ⁇ HyColl-RGD in Na 2 C0 3 and NaHCOs (pH 10).
  • FIG 40 A transparent HyColl hydrogel (20% w/w) fabricated by chemically crosslinking using a zero length cross linker. Thermal stability of 20% (w/w) is 61 °C.
  • HyColl hydrogel as a function of frequency ( ⁇ ) at fixed strain amplitude ( ⁇ 0 0.1 %).
  • FIG. 46 A Gelcode blue stained SDS-PAGE gel showing the purification of HyColl- Tyrosine-RGDS.
  • the lanes contain the following: Lane 1 : Precision plus proteinTM standards; Lane 2: Purified HyColl-Tyrosine-RGDS.
  • FIG. 47 CD wavelength scans of 10 ⁇ HyColl-Tyrosine-RGDS solutions showing an ellipticity maximum at 220nm and a minimum at 198 nm characteristic of triple helix secondary structure in acidic, neutral, and basic pH buffers.
  • FIG 48 CD thermal scans of HyColl-Tyrosine-RGDS in citric acid and Na 2 HP0 4 (pH 4) showing the denaturation with increasing temperature (triangles) and refolding of the protein with decreasing temperature (dots).
  • FIG. 49 CD thermal scans of 10 ⁇ HyColl-Tyrosine-RGDS in NaH 2 P0 4 and Na 2 HP0 4 (pH 7) showing the denaturation with increasing temperature (triangles) and refolding of the protein with decreasing temperature (dots).
  • Figure 50 CD thermal scans of 10 ⁇ HyColl-Tyrosine-RGDS in PBS (pH7.4) showing the denaturation with increasing temperature (triangles) and refolding of the protein with decreasing temperature (dots).
  • FIG 51 CD thermal scans of 10 ⁇ HyColl-Tyrosine-RGDS in NaHC0 3 and Na 2 C0 3 (pH 10) showing the denaturation with increasing temperature (triangles) and refolding of the protein with decreasing temperature (dots).
  • Figure 52 Micro-DSC heating and cooling scan for 100 ⁇ HyColl-Tyrosine-RGDS in Na 2 HP0 4 and citric acid (pH 4).
  • FIG. 53 Micro-DSC heating and cooling scan for 100 ⁇ HyColl-Tyrosine-RGDS in Na 2 HP0 4 and NaH 2 P0 4 (pH 7).
  • FIG. 54 Micro-DSC heating and cooling scan for 100 ⁇ HyColl-Tyrosine-RGDS in
  • FIG. 55 Micro-DSC heating and cooling scan for 100 ⁇ HyColl-Tyrosine-RGDS in Na 2 C0 3 and NaHC0 3 (pH 10).
  • FIG. 56 C2C12 cells on 25uM Hycoll(a) and Hycoll-RGD(b) solution coated PS surface.
  • FIG. 58 WST-1 assay on 25uM PS surface coated with different percentage of HyColl-Tyrosine-RGDS.
  • Figure 60 Fluorescence Spectrum for HyColl-Tyrosine-RGDS Photo-crosslinked hydrogel using SPS only (blue light exposure). Excitation at 315 nm.
  • FIG. 62 HyColl-Tyrosine-RGDS Photo-crosslinked hydrogel using (a) 48 U HRP and (b) 24U HRP.
  • Figure 63 Fluorescence Spectrum for HyColl-Tyrosine-RGDS Enzyme crosslinked hydrogel using HRP. Excitation at 260 nm.
  • Figure 64 Stained C2C12 cells on 10% photo cross-linked Hycoll-Tyrosine-RGDS hydrogel.
  • Figure 65 Stained C2C12 cells on 10% enzyme cross-linked Hycoll-Tyrosine-RGDS hydrogel.
  • sub-domain B a sub-domain sequence of the CL domain from Scl2 referred to as sub-domain B (Yu, Z, Brodsky, B., and Inouye, M. (201 1) J. Biol. Chem 286, 18960-18968) was chosen and modified as described herein. Modification of the native sequence involved amino acid substitution to increase the availability of key functional groups (e.g. primary amines, aromatics, and/or glutamine) for crosslinking purposes and to change the overall charge distribution of the sequence so that it is highly soluble at elevated concentrations in aqueous solutions at acidic, physiologic and basic pH.
  • key functional groups e.g. primary amines, aromatics, and/or glutamine
  • This sequence can be repeated sequentially to make a novel stable sequence that can be fused with the known N-terminal V domain and expressed in bacteria ( Figure 1). Controlling the number of repeats of the sequence allows control of the overall charge, and therefore solubility can be maintained over a broad pH range even if charged sequences are added to the sequence. This in part makes the material described herein particularly well-suited to bio-printing.
  • the molecular weight (MW) for the sequence shown in Figure 1 is 32 kDa.
  • the iso-electric point (pi) is estimated to be 5.6.
  • the total number of negatively charged residues is 52.
  • the total number of positively charged residues is 42.
  • the atomic composition is as follows:
  • a protein that is highly soluble at elevated concentrations across a broad pH range, including physiologic pH provides to those skilled in the art numerous approaches to stabilize the protein to produce not only sponges and foams but a wide array of thermally stable transparent hydrogel materials that can be tuned to possess a variety of physical and biological properties for numerous applications.
  • cell binding integrins such as RGDS and integrins with spacer sequences such as GSTSGSGT designed to improve integrin bioavailability (Figure 2) can be inserted into the sequence to allow for control of cell growth and differentiation.
  • Other cell binding and signalling domains such as DGEA, REDV, YIGSR, IKVAV, KAFAK, and VAPG ( Figure 3) as well as biomineralization domains (e.g. for hydroxyapatite) and biomimetic structural proteins (e.g. elastin, resilin, abductin) may be inserted.
  • Spacer sequences can be modified and spacer lengths can be extended. The length of the triple helix can be varied.
  • Modification of the charge distribution for instance by combining more than one type of the B-domain collagen-mimetic motif may allow the promotion of self-assembly of the material.
  • Collagen-like sequences can be produced with and without the non-collagenous V domain.
  • this well-defined collagen-like material can be produced with high purity and with no risk of contamination by the infectious agents associated with animal sourced collagen (e.g. bovine spongiform encephalopathy, ovine and caprine scrapie, and other zoonoses).
  • the sequence can be modified to enhance or change specific biological or functional properties.
  • modification of the native amino acid sequence to increase the availability of key functional groups (e.g.
  • composition consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1 % by weight of non-specified components.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • terapéuticaally effective amount means a quantity sufficient, when administered to a subject, including a mammal, for example a human, to achieve a desired result.
  • Effective amounts of the compounds described herein may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage or treatment regimes may be adjusted to provide the optimum therapeutic response, as is understood by a skilled person.
  • a treatment regime of a subject with a therapeutically effective amount may consist of a single administration, or alternatively comprise a series of applications.
  • the length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the subject, the concentration of the agent, the responsiveness of the patient to the agent, or a combination thereof.
  • the effective dosage of the agent used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.
  • the antibodies described herein may, in aspects, be administered before, during or after treatment with conventional therapies for the disease or disorder in question, such as cancer.
  • subject refers to any member of the animal kingdom, typically a mammal.
  • mammal refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is human.
  • Administration "in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • pharmaceutically acceptable means that the compound or combination of compounds is compatible with the remaining ingredients of a formulation for pharmaceutical use, and that it is generally safe for administering to humans according to established governmental standards, including those promulgated by the United States Food and Drug Administration.
  • pharmaceutically acceptable carrier includes, but is not limited to solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and/or absorption delaying agents and the like.
  • an "isolated" biological component (such as a protein) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., chromosomal and extra-chromosomal DNA and RNA, other proteins and organelles.
  • Proteins and peptides that have been "isolated” include proteins and peptides purified by standard purification methods. The term also includes proteins and peptides prepared by recombinant expression in a host cell, as well as chemically synthesized proteins and peptides.
  • Activity refers to a biological activity of the recombinant collagen-like polypeptides described herein, wherein “biological” activity refers to a biological function (either inhibitory or stimulatory) caused by the recombinant collagenlike polypeptides.
  • biologically active or “biological activity” when used in conjunction with
  • recombinant collagen-like polypeptide means recombinant collagen-like polypeptide or fragment thereof that exhibits or shares an effector function of a collagen-like polypeptide or collagen.
  • “Variants” are biologically active polypeptides or fragments thereof having an amino acid sequence that differs from the sequence of a recombinant collagen-like polypeptide described herein, by virtue of an insertion, deletion, modification and/or substitution of one or more amino acid residues within the comparative sequence. Variants generally have less than 100% sequence identity with the comparative sequence.
  • a biologically active variant will have an amino acid sequence with at least about 70% amino acid sequence identity with the comparative sequence, such as at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the variants include peptide fragments of at least 10 amino acids that retain some level of collagen-like polypeptide or collagen activity.
  • Variants also include polypeptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the comparative sequence. Variants also include polypeptides where a number of amino acid residues are deleted and optionally substituted by one or more amino acid residues. Variants also may be covalently modified, for example by substitution with a moiety other than a naturally occurring amino acid or by modifying an amino acid residue to produce a non-naturally occurring amino acid.
  • a substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in
  • amino acid residues may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity).
  • a conservative mutation may be an amino acid substitution.
  • Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group.
  • basic amino acid it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH.
  • Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K).
  • neutral amino acid also “polar amino acid”
  • hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gin or Q).
  • hydrophobic amino acid (also “non-polar amino acid”) is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (lie or I),
  • phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).
  • Acidic amino acid refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).
  • Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA- N, or any other appropriate software that is known in the art.
  • the substantially identical sequences of the present invention may be at least 85% identical; in another example, the substantially identical sequences may be at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% (or any percentage there between) identical at the amino acid level to sequences described herein. In specific aspects, the substantially identical sequences retain the activity of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s).
  • the collagen-like polypeptide or fragment thereof described herein may also comprise additional sequences to aid in its expression, detection, or purification. Any such sequences or tags known to those of skill in the art may be used.
  • the collagen-like polypeptide or fragment thereof may comprise a targeting or signal sequence (for example, but not limited to ompA), a detection tag, exemplary tag cassettes include Strep tag, or any variant thereof; see, e.g., U.S. Patent No.
  • His tag Flag tag having the sequence motif DYKDDDDK, Xpress tag, Avi tag, Calmodulin tag, Polyglutamate tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Softag 1 , Softag 3, V5 tag, CREB-binding protein (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), green fluorescent protein (GFP), Thioredoxin tag, or any combination thereof; a purification tag (for example, but not limited to a His 5 or His 6 ), or a combination thereof.
  • CBP CREB-binding protein
  • GST glutathione S-transferase
  • MBP maltose binding protein
  • GFP green fluorescent protein
  • Thioredoxin tag Thioredoxin tag
  • the additional sequence may be a biotin recognition site such as that described by Cronan et al in WO 95/04069 or Voges et al in WO/2004/076670.
  • linker sequences may be used in conjunction with the additional sequences or tags.
  • nucleic acids encoding the collagen-like polypeptides or fragments thereof may be provided in expression vectors operably linked to an expression sequence, a promoter and an enhancer sequence.
  • a variety of expression vectors for the efficient synthesis of polypeptides in prokaryotic, such as bacteria and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems have been developed.
  • the vectors described herein can comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • prokaryotic cloning vectors include plasmids from E. coli, such as colEI, pCRI, pBR322, pMB9, pUC, pKSM, and RP4.
  • Prokaryotic vectors also include derivatives of phage DNA such as MI3 and other filamentous single-stranded DNA phages.
  • An example of a vector useful in yeast is the 2 ⁇ plasmid.
  • Suitable vectors for expression in mammalian cells include well-known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences and shuttle vectors derived from combination of functional mammalian vectors, such as those described above, and functional plasmids and phage DNA.
  • Additional eukaryotic expression vectors are known in the art (e.g., P J. Southern & P. Berg, J. Mol. Appl. Genet, 1 :327-341 (1982); Subramani et al, Mol. Cell. Biol, 1 : 854-864 (1981); Kaufinann & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol, 159:601 -621 (1982); Kaufhiann & Sharp, Mol. Cell.
  • the expression vectors contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed.
  • the control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence.
  • useful expression control sequences are the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.
  • Cell lines of typical use are selected based on high level of expression, constitutive expression of protein of interest and minimal contamination from host proteins.
  • Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines, such as but not limited to, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells and many others. Suitable additional eukaryotic cells include yeast and other fungi.
  • Useful prokaryotic hosts include, for example, E. coli, such as E. coli BL-21 , E. coli SG-936, E. coli HB 101 , E. coli W31 10, E. coli X1776, E. coli X2282, E. coli DHI , and E. coli MRC1 , Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.
  • present recombinant host cells can be used to produce polypeptides by culturing the cells under conditions permitting expression of the polypeptide and purifying the polypeptide from the host cell or medium surrounding the host cell.
  • Targeting of the expressed polypeptide for secretion in the recombinant host cells can be facilitated by inserting a signal or secretory leader peptide-encoding sequence (See, Shokri et al, (2003) Appl Microbiol Biotechnol. 60(6): 654-664, Nielsen et al, Prot. Eng., 10: 1 -6 (1997); von Heinje et al., Nucl.
  • secretory leader peptide elements can be derived from either prokaryotic or eukaryotic sequences.
  • secretory leader peptides are used, being amino acids joined to the N-terminal end of a polypeptide to direct movement of the polypeptide out of the host cell cytosol and secretion into the medium.
  • the collagen-like polypeptides and fragments thereof described herein can be fused to additional amino acid residues.
  • amino acid residues can be bioactive domains or assembly domains, such as the V domain or a foldon sequence such as
  • HyCoN and “HyColl-RGD” describe the specific sequences of the collagen-like polypeptides shown in Figures 1 and 2A. While these specific polypeptides were made and used in the examples, it will be understood that the polypeptides, compositions, and methods described herein are not limited to HyColl and HyColl-RGD and are more broadly applicable to any modified collagen-like protein that is more soluble in acidic, neutral, and/or basic solutions than a non-modified collagen like protein or fragment thereof, that is stable in solution from about 5 to about 36°C at physiologic pH, such as from about 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 1 1 °C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C,
  • modified B subdomains of collagen-like proteins are derived from bacteria and do not have attributes that make them useful as a collagen replacement.
  • modifying the B subdomain of the collagen-like protein to increase the availability of lysines, and/or tyrosines and/or tryptophans and/or phenylalanines and/or glutamines and/or histidines for crosslinking and/or to change the overall charge distribution of the sequence so that it is highly soluble at elevated concentrations results in a collagenlike protein that finds use in a number of applications, including bio-inks, ophthalmic devices, wound dressings, and so on. Cysteines may be inserted at the terminal ends to facilitate conjugation with other polymers and/or peptides.
  • the absolute number or position of the modified amino acids is not critical. As few as one amino acid may be replaced with a lysine, tyrosine tryptophan, phenylalanine, histidine and/or glutamine, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitutions may be made.
  • polypeptide comprising a sequence with at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, or 99%) sequence identity to the sequence of: MNHKVHMHHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLT YLQEREQAENSWRKRLLKGIQDHALDGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGE QGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGKDGERGPVGPAGPKGEQGPQGL PGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGK DGERGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGK DGERGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGK D
  • MNHKVHMHHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLT YLQEREQAENSWRKRLLKGIQDHALD or a fragment or variant thereof is cleaved from the polypeptide after expression.
  • This sequence represents the V domain of a collagen-like peptide.
  • moieties include, but are not limited to peptides, carbohydrates, small molecules, drugs, antibodies, PEG-based compounds, toxins, dyes, imaging agents or binding sequences.
  • polypeptides described herein may be engineered to include a sequence that facilitates binding of the polypeptide to a targeted cell type or provides a natural cleavage sited for degradation in the body.
  • Binding sequences include for example, integrin binding domains such as those identified for ⁇ 2 ⁇ 1 integrin or an ⁇ 3 ⁇ 1.
  • Other sequences include the known type II collagen binding site for DDR2.
  • Cleavage sequences may include, but are not limited to, one or more sequences within the family of Matrix Metalloproteinase (MMP) domains, e.g. MMP-1 , MMP-2, MMP-8, MMP-13 and MMP-18 which cleave type I, II and III collagens and MMP-2 and MP-9 which cleave denatured collagens.
  • MMP Matrix Metalloproteinase
  • the polypeptide may comprise a bioactive domain, such as a cell binding domain, a cell signalling domain, a biomineralization domain, a metallization domain, an elastomeric domain, and/or a biomimetic structural protein.
  • a bioactive domain such as a cell binding domain, a cell signalling domain, a biomineralization domain, a metallization domain, an elastomeric domain, and/or a biomimetic structural protein.
  • the bioactive cell binding domain is linked to the C-terminal end of the polypeptide with an optional linker sequence.
  • the linker sequence comprises either a short sequence such as (GSTSGSGT)n, wherein n is an integer or a long sequence such as
  • the cell binding comprises at least one of RGD, RGDS, DGEA, REDV, YIGSR, IKVAV, KAFAK, and VAPG
  • the biomineralization domain comprises at least one of MLPHHGA, SVSVGMKPSPRP, ESQES, and QESQSEQDS
  • the metallization domain comprises at least one of NPSSLFRYLPSD, MHGKTQATSGTIQS, AQNPSDNNTHTH and RLELAIPLQGSG
  • the elastomeric domain comprises at least one of VPGVG, (AG) 3 EG, GYSGGRPGGQDLG, GGFGGMGGGS, PGQGQQ, and GYYPTSPQ.
  • the polypeptide is modified in aspects to include cross-linking elements such as, for example, lysine, tyrosine, tryptophan, phenylalanine, glutamine, histidine, and combinations thereof.
  • One or more terminal cysteines for conjugation to other polymers and/or peptides may be included and, in aspects, the polypeptide is ionically crosslinked for example with cationic and/or anionic crosslinkers, such as spermidine or tri-poly-phosphate respectively.
  • cationic and/or anionic crosslinkers such as spermidine or tri-poly-phosphate respectively.
  • fragment of the polypeptide described herein may comprise the sequence
  • each X is independently selected from lysine, tyrosine, tryptophan, phenylalanine, histidine, and glutamine.
  • fragment of the polypeptide described herein may comprise the sequence
  • GPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPR GERGEAGPAGKDGERGPVGPA or a sequence having at least 70% sequence identity thereto, wherein at least one amino acid residue is substituted with a lysine, a tyrosine, a tryptophan, a phenylalanine, a histidine, or a glutamine.
  • fragment of the polypeptide described herein may comprise the sequence
  • GPXGEQGPQGLPGKDGEAGAQGPAGPMGPAGEXGEKGEPGYXGAKGDRGETGPXGPX GERGEAGPAGKDGERGPVGPA or a sequence having at least 70% sequence identity thereto, wherein each X is independently selected from lysine, tyrosine, tryptophan, phenylalanine, histidine, and glutamine.
  • sequences may be repeated multiple times, such that there are 1 , 2, 3, 4, 5, 6,
  • the polypeptide described herein may include a V domain of a collagenlike protein, which may be from the same species as the modified polypeptide or of a different species, or it may be an artificial sequence.
  • the V domain may be designed to be cleaved following expression leaving behind the sequence described above.
  • the polypeptides described herein typically encode a modified collagen-like protein or fragment thereof.
  • the modified collagen-like protein has attributes that are improved as compared to the corresponding non-modified or wild-type collagen-like protein, such as increased solubility in acidic, neutral, and/or basic solutions, stability in solution from about 5 to about 36°C at physiologic pH, ability to self-assemble into a triple helix in acidic, neutral, and/or basic solutions, thermal stability that is about the same or better than collagen, and increased collagenase-resistance as compared to collagen.
  • polypeptides encoding a modified collagen-like protein or fragment thereof described herein may be modified to increase availability of lysines, and/or tyrosines and/or tryptophans and/or phenylalanines and/or glutamines and/or histidines for crosslinking.
  • polypeptides described herein may be fused to other polypeptides and produced as fusion proteins. Typically, the polypeptides described herein are fused to other parts of a collagen-like protein, such as the remainder of the CL domain and/or the V domain. Thus, also provided herein is a modified collagen-like protein comprising the polypeptide or fragment thereof described herein.
  • Collagen-like proteins are generally derived from bacteria, such as Streptococcus pyogenes and the specific HyColl sequence shown in Figure 1 herein is a modified Scl2 protein, wherein a naturally occurring portion of the Scl2 protein is substituted with the polypeptide shown in Figure 1.
  • the collagen-like protein is typically a V-CL protein comprising an N-terminal globular V domain, followed by a collagen-like CL domain, wherein the CL domain comprises or consists of at least one copy of the polypeptide described herein, which may be present in a single copy or may be repeated one or many times, either substantially consecutively, consecutively, or with intervening sequences of any length.
  • collagen-like proteins may be derived from sources that include S. pyogenes, Methylobacterium sp4-46, Solibacter usitatus, Streptococcus equi ScIC, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Rhodopseudomonas palustris,
  • Streptococcus pneumoniae A Corynebacterium diphtheria, Actinobacteria (e.g.,
  • Nocardioides species Rubrobacter xylanophilus, Salinispora arenicola, Salinispora tropica, and Streptomyces species
  • Alphaproteobacteria e.g., Anaplasma species
  • Methylobacterium radiotolerans Nitrobacter winogradskyi, Paracoccus denitrificans, Rhizobium leguminosarum, Rhodobacter sphaeroides, Rhodopseudomonas palustris, Sphingomonas wittichii, and Wolbachia species), Bacteroidetes (e.g., Bacteroides thetaiotaomicron), Betaproteobacteria (e.g., Azoarcus species, Burkholderia ambifaria, Burkholderia cenocepacia, Burkholderia phymatum, Burkholderia vietnamiensis,
  • Bacteroidetes e.g., Bacteroides thetaiotaomicron
  • Betaproteobacteria e.g., Azoarcus species, Burkholderia ambifaria, Burkholderia cenocepacia, Burkholderia phymat
  • Cyanobacteria e.g., Cyanothece species, Synechocystis species, Trichodesmium erythraeum
  • Deinococcus e.g., Deinococcus radiodurans
  • Deltaproteobacteria e.g., Anaeromyxobacter
  • Epsilonproteobacteria e.g., Campylobacter curvus
  • Firmicutes e.g., Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Bacillus subtilis, Clostridium botulinum, Clostridium phytofermentans, Enterococcus faecalis, Geobacillus kaustophilus, Lactobacillus casei, Lactobacillus plantarum, Lactococcus lactis, Lysinibacillus sphaericus, Staphylococcus haemolyticus, Streptococcus agalactiae, and Streptococcus pneumoniae), and Gammaproteobacteria (e.g., Citrobacter koseri, Enterobacter species, Escherichia coli, Klebsiella pneumoniae, Legionella pneumophila, Photorhabdus luminescens, Pseudom
  • cryohalolentis Saccharophagus degradans, Salmonella enterica, Salmonella typhimurium, Serratia proteamaculans, Shewanella amazonensis, Shewanella baltica, Shewanella frigidimarina, Shewanella halifaxensis, Shewanella loihica, Shewanella oneidensis,
  • Shewanella pealeana Shewanella putrefaciens, Shewanella sediminis, Shewanella woodyi, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, and Vibrio harveyi).
  • the collagen-like proteins for use herein may be sub-sequences from any Streptococcal collagen-like (Scl) protein e.g. Scl 1. (Xu et al. (2002) J. Biol. Chem. 277, 27312-27318); sub-sequences from any Bacillus collagen like protein (Bel) e.g. Bel 1 or a sequence known as FZB42 (Pizarro-Guajardo et al. (2014) Anaerobe 25, 18-30; Zhao et al. (2015) PLOS February 6, pp 1-16); sub-sequences from any Pasteuria collagen-like protein (Pel) e.g. Pel 1 a (Mouton et al.
  • Scl Streptococcal collagen-like protein
  • the polypeptides described herein may be encoded by nucleic acid molecules, which may be degenerate.
  • the nucleic acid molecules may be in expression vectors, which may themselves be expressed by a recombinant host cell of any species, as is well understood by a skilled person.
  • compositions may be formulated into compositions and those compositions are generally transparent.
  • the compositions may be crosslinked and may be in the form of a hydrogel, which may also be transparent.
  • the compositions may be, for example, sponges or foams or they may be thin films that can be applied to a wound or used in the eye.
  • the compositions optionally comprise additional components such as a pharmaceutically acceptable carrier, diluent, and/or buffer.
  • the polypeptides and compositions described herein in aspects can also be used to fabricate complex material shapes and bio-print three-dimensional constructs.
  • the collagen-like polypeptides described herein can easily be fabricated into complex material shapes and combined with other elements (e.g. cells, growth factors, nanoparticles, other proteins and polymers) using conventional methods, including but not limited to solution casting, molding, bioprinting, and electrospinning. Stabilization (i.e.
  • gelation of the construct can occur, for example, through chemical, physical, ionic, photo, or enzyme based crosslinking methods to tune the properties of the fabricated collagen-like polypeptide construct.
  • High solubility at acidic, neutral and basic pH allows for a much broader range of approaches to be used compared to collagen and other collagen-like peptides.
  • high concentration collagen-like polypeptide solutions can be crosslinked at acidic and neutral pH to fabricate thermally stable, transparent hydrogels.
  • targets e.g., primary amines, aromatics, and/or glutamine
  • the user can tune the physical properties (e.g. tensile strength, modulus) of the crosslinked hydrogel.
  • a chemical crosslinker such as EDC or DMTMM
  • the physical properties e.g. tensile strength, modulus
  • These hydrogels are easily prepared by using a syringe based system that facilitates mixing of the collagen-like polypeptide solution and the crosslinking agent(s). This solution is then dispensed from the system and can be cast as flat sheets or in molds to give a desired shape and thickness.
  • modification of the collagen-like polypeptide sequence with tyrosine and/or tryptophan and/or phenylalanine allows chemical, enzyme and photo-crosslinking (e.g. visible and ultraviolet light) approaches to be used.
  • a metal-ligand complex such as ruthenium-tris(2,2'- bipyridyl) dichloride in conjunction with an electron acceptor inter- and intra-di-tyrosine bonds can be created through a mechanism which does not involve the formation of potentially detrimental species such as singlet oxygen, superoxide or hydroxyl radicals ( U.S. Patent Application Publication No. 201 1/001908, which is incorporated by reference herein in its entirety).
  • a photo-initiator such as riboflavin in the presence of visible or UV light can be used to form di-tyrosine complexes to fabricate thermally stable hydrogels.
  • the degree of photo-crosslinking is a function of the time and energy of the irradiation, as well as the final concentrations of the cross-linking agents, the degree of cross-linking can be varied to achieve the properties required for a specific application.
  • Enzyme catalyzed crosslinking of the aromatic amino acids can also be used to fabricate hydrogels. For example, it has been previously demonstrated that horseradish peroxidase can be used to form di-tyrosine bonds (Partlow, BP., et al.(2014) Adv Fund Mater.
  • the same solution can be bio-printed and crosslinked to fabricate 3-D layered constructs ranging in size, shape and thickness. Given their relatively low viscosities, collagen-like polypeptide bio-inks are associated with low shear stress during the printing process and therefore should improve the viability of printed cells.
  • the preparation of high concentration collagen-like polypeptide bio-ink solutions can be used to facilitate the bio- printing of structures with high shape fidelity.
  • Electrospinning is a fabrication process that uses an electric field to control the deposition of polymer or protein fibers onto a target substrate to fabricate complex and seamless three dimensional shapes (Matthews, J, Wnek, G., Simpson, D., and Bowlin, G. (2002) Biomacromolecules 3, 232-238).
  • the presence of charge, along with the combination of high solubility and low viscosity, provide collagen-like polypeptide solutions with the potential to be used successfully to fabricate electrospun scaffolds for a variety of tissue engineering applications.
  • the collagen-like polypeptides described herein may find application in a variety of biomedical and regenerative medicine fields such as but not limited to cosmetics, cosmetic surgery materials, dermal implants, coatings, cell encapsulation, cell based assays, drug delivery, wound therapy and tissue implants such as artificial skin.
  • the collagen-like polypeptides described herein have been used as shown in Figure 40 (see Example 7 below) to produce thermally stable, transparent hydrogels which is important for ophthalmic device applications such as corneal implants and implants designed for refractive index correction.
  • the collagen-like polypeptides described herein may also find applications in urinary incontinence products, as well as protein dietary supplements, carriers, food additives, edible films and coatings for food and beverages.
  • EDC Ethyl-3-(3-dimethyl aminopropyl) carbodiimide
  • DTMM 4,6-dimethoxy-1 ,3,5-triazin- 2-yl)-4-methylmorpholinium chloride
  • NaHC0 3 sodium bicarbonate
  • ruthenium- tris(2,2'-bipyridyl) dichloride sodium persulfate, ammonium persulfate, horse radish peroxidase, hydrogen peroxide, sodium carbonate (Na 2 C0 3 ) and collagenase (type I
  • Clostridium histolyticum were supplied by Sigma-Aldrich (Oakville, Ontario, Canada). N- hydroxysuccinimide (NHS) was supplied by Fluka (Buchs, Switzerland). Citric acid (CeHsCv), 2-(4-morpholino)-ethane sulfonic acid (MES), sodium phosphate dibasic (Na 2 HP0 4 ), and sodium phosphate monobasic (NaH 2 P0 4 ) were supplied by Fischer Scientific (Nepean, Ontario, Canada). All other reagents were of analytical grade and used as received.
  • Phosphate buffer was prepared from tablet form supplied by MP Biomedicals (Santa Ana, California, USA).
  • DNA Oligos were purchased from Integrated DNA Technologies (Illinois, USA) and GeneScript (Piscataway, NJ, USA). Untreated polystyrene petri dishes were purchased from Corning Inc. (NY, USA). C2C12 mouse myoblast cells were purchased from ATCC
  • DMEM medium fetal bovine serum (FBS) and streptomycin/penicillin (P/S) were purchased from Fisher Scientific (Ottawa, ON, Canada).
  • the WST-1 Cell Proliferation Assay was purchased from Roche (Laval, QC, Canada). LIVE/DEADTM
  • Viability/Cytotoxicity Kit for mammalian cells were purchased from ThermoFisher (Ottawa, ON, Canada). All polymerases, enzymes, and buffers for DNA ligation, digestion and mutation were purchased from New England Biolabs. All other chemicals were purchased either from Sigma-Aldrich or Fisher Scientific (Ottawa, ON, Canada). Methods
  • HyColl Recombinant bacterial collagen-like sequence
  • HyColl construct (including HyColl and other HyColl-based proteins) was expressed in E. Coli. Strain BL-21. Protein expression was performed both in small and large scales. Small scale expression in shake flasks was carried out at 37°C in 1 L of SOB media containing 100 ⁇ g/ml ampicillin, and induced with isopropyl-D-thiogalactoside (IPTG) (final concentration 1 mM) overnight at 16°C when the optical density at 600 nm (OD600) approached 0.8. Cells were then harvested by centrifugation at 8000 g for 15 minutes at 4°C.
  • IPTG isopropyl-D-thiogalactoside
  • a feed solution containing glucose (500 g/L), yeast extract (100 g/L), MgSC - 7H20 (15 g/L) and ampicillin (100 mg/L) was automatically added into the fermentor as needed to maintain pH neutral after fermentation. Cultures were induced by IPTG overnight at 16°C when the OD600 reached approximately 15. Cells were then harvested by centrifugation at 8000 g for 30 minutes at 4°C.
  • the resulting lysate was cleared by centrifugation at 20,000rpm for 20 min at 4°C and the supernatant was further passed through a 0.22 ⁇ filter membrane before purifying with columns packed with IMAC SepharoseTM 6 Fast Flow (GE Healthcare Life Sciences) charged with Ni 2+ ions either in a gravity flow column or via an FPLC system. Cleared lysate was loaded onto columns pre-equilibrated with binding buffer, washed with 3 column volume of binding buffer, and then eluted with 3 column volume of elute buffer (20mM Na 2 HP04/NaH2P04 pH 7.4, 500mM NaCI and 500mM imidazole). The purified protein was checked by SDS-PAGE and GelCode blue staining.
  • HyColl, HyColl-RGD or porcine collagen solution were weighed and mixed. Calculated volumes of aqueous crosslinking solutions were added to their respective solutions at 4 - 6°C and thoroughly mixed. The final solution was cured as either a flat sheet or in small tubes as required. Hydrogels were prepared using EDC in the presence of NHS and DMTMM respectively. Crosslinking ratios throughout are defined as the molar equivalent ratio of 8-amine (NH2): EDC or DMTMM.
  • HyColl solutions prepared in PBS (pH 7.4) were bio-printed in layers using a custom designed bio-printer equipped with dual piston extrusion for precise: XYZ positioning, deposition rate and temperature control. Sample layer height control and sample surface characterization was performed using a custom-designed contact metrology capability. The printed material was crosslinked using the above method.
  • Pre-determined amounts of lyophilized HyColl-Tyrosine-RGDS or HyColl were weighed and mixed in 0.1 M PBS. A protein concentration in the range of 0.5% (w/v) to greater than 30% (w/v) can be used. Calculated volumes of aqueous crosslinking solutions (e.g. Ru(bpy)3C , SPS, APS) were added to their respective protein solutions at room temperature and thoroughly mixed. Solutions were exposed for 30 seconds to a 455 nm LED light source providing 28 mW/cm 2 to the sample.
  • aqueous crosslinking solutions e.g. Ru(bpy)3C , SPS, APS
  • sample final crosslinking reagent molarities are provided in Table 1.
  • the final solution was cured as either a flat sheet or in small tubes as required.
  • HyColl-Tyrosine-RGDS solutions prepared in PBS (pH 7.4) were bio-printed in layers using a custom designed bio-printer equipped with dual piston extrusion for precise: XYZ positioning, deposition rate and temperature control. Sample layer height control and sample surface characterization was performed using a custom-designed contact metrology capability. The printed material was crosslinked using the above method. Table 1. Sample volumes for a Photo-Crosslinked HyColl-Tyrosine-RGDS solution.
  • HyColl-Tyrosine-RGDS or HyColl were weighed and mixed in 0.1 M PBS.
  • the solution concentration in the range of 0.5% (w/v) to greater than 30% (w/v) can be used.
  • Calculated volumes of aqueous crosslinking solutions e.g. HRP, hydrogen peroxide 30%
  • HRP hydrogen peroxide 30%
  • Sample layer height control and sample surface characterization was performed using a custom-designed contact metrology capability.
  • the printed material was crosslinked using the above method.
  • Agilent 1 100 capillary-HPLC system (Agilent Technologies, Santa Clara, CA) is hooked up with LTQ-Orbitrap mass spectrometer (Thermo Electron, Waltham, MA).
  • the solvent system consists of buffer A of 0.1 % FA in water, and buffer B of 0.1 %FA in acetonitrile.
  • Dried down protein digest were acidified with 0.5% (v/v) formic acid and loaded on a 75 ⁇ I.D. ⁇ 100 mm fused silica analytical column packed in-house with 3 ⁇ ReproSil-Pur C18 beads (100 A; Dr. Maisch GmbH, Ammerbuch, Germany) at a flow rate of 1 .5 ⁇ _/ ⁇ for 15min.
  • An LTQ-Orbitrap mass spectrometer (ThermoFisher Scientific, San Jose, CA) equipped with a nano-electrospray interface was operated in positive ion mode.
  • the spray voltage was set to 2.0 kV and the temperature of heated capillary was 200°C.
  • the instrument method consisted of one full MS scan from 300 to 1700 m/z.
  • R 60,000
  • MW is the molecular weight of protein and z is the charge state.
  • CD spectra were measured for 10 ⁇ aqueous HyColl, HyColl-RGD and HyColl- Tyrosine-RGDS solutions in citric acid/Na 2 HP0 4 buffer at pH 4, citric acid/Na 2 HP0 4 buffer at pH 7, Na 2 HP04/NaH 2 P04 buffer at pH 7, NaHC0 3 /Na 2 C0 3 buffer at pH 10, 50 mM MES at pH 4.5 and PBS (pH 7.4) in a 1 mm path length quartz cuvette using a Jasco 815 spectropolarimeter (Jasco Inc., Easton, USA) to evaluate secondary structure, melting temperature and refolding properties.
  • Jasco 815 spectropolarimeter Jasco Inc., Easton, USA
  • the CD signals were collected as a function of wavelength (from 185nm to 250nm) at constant temperature (20°C) to determine the protein secondary structure and as a function of temperature for a selected wavelength (e.g. 220 nm). Wavelength scans were also conducted for the protein at pH 7 (citric acid and Na 2 HP0 4 buffer) from 185 nm to 250 nm by increasing the temperature from 20°C to 50°C followed by decreasing the temperature to 5°C. Ten scans were recorded and averaged for each sample. In a temperature scan, the CD signal was collected as a function of temperature for a selected wavelength.
  • HyColl, HyColl-RGD and HyColl-Tyrosine-RGDS solution were studied by monitoring the CD signal at 220 nm as a function of temperature from 15 to 55°C at a rate of 1 °C/min, and the refolding ability was studied by monitoring the CD signal at 220 nm as a function of temperature from 55 to 15°C at a rate of 1 °C/min.
  • concentration in M is the path length in cm, and n is the number of amino acid residues in a protein.
  • FTIR spectra were recorded on a Nicolet Nexus 6700 FTIR in the 1300 - 1800 cnr 1 wavelength range. All samples were prepared in deuterated water. The spectrum was deconvoluted using a best fit Gaussian model (i.e. LMFIT/Python).
  • spectrophotometer Biotek Instruments, Vermont USA equipped with temperature control. Di-tyrosine fluorescence was measured using excitation/emission wavelengths of 315/410 nm, respectively. Tyrosine fluorescence was measured using excitation/emission wavelengths of 260/305 nm, respectively.
  • RGDS solutions at pH 4, pH 7 and pH 10 and 10 HyColl and HyColl-RGD solution at pH 4, pH 7 and pH 10 using a nano-DSC III calorimeter (Calorimetry Sciences, Lindon, USA).
  • the heat rate scan was recorded at the rate of 1 °C/min as temperature increased from 15 to 55°C. Data was then analyzed with the software package Cpcalc (Calorimetry Sciences, Lindon, USA) to determine the denaturation temperature and the dependence of heat capacity on temperature.
  • the thermal stability of the HyColl, HyColl-RGD and HyColl-RGD-Tyrosine hydrogels were examined using a Q2000 differential scanning calorimeter (TA Instruments, New Castle, DE). Heating scans were recorded within the range of 8 to 80°C at a scan rate of 5°C mirr . Pre-weighed samples of the PBS-equilibrated hydrogels (weights ranging from 5 to 10 mg) were surface-dried with filter paper and hermetically sealed in an aluminum pan to prevent water evaporation. A resulting heat flux versus temperature curve was then used to calculate the denaturing temperature (T d ). The denaturing temperature is given by T ma x of the endothermic peak.
  • the tensile strength, Young's moduli and elongation at break of the EDC/NHS HyColl crosslinked hydrogels were determined on an Instron electromechanical universal tester (Model 3342) equipped with Series IX/S software, using a crosshead speed of 10 mm min -1 and a gauge length for testing of 5 mm. 0.55mm hydrogels were equilibrated in PBS and cut into 10 mm ⁇ 5 mm rectangular sheets. A minimum of three specimens were measured for each hydrogel formulation. Dynamic shear moduli of the same crosslinked hydrogels were measured in strain-controlled oscillatory shear deformation using a Paar Physica rheometer (MCR 301) in a parallel plate geometry.
  • MCR 301 Paar Physica rheometer
  • C2C12 mouse myoblast cells were cultured in a humidified incubator at 37 °C and
  • HyColl and Hycoll-Tyrosine-RGDS were also examined for several combinations of HyColl and Hycoll-Tyrosine-RGDS including 100% HyColl and 0% HyColl-Tyrosine-RGDS, 95% HyColl and 5% HyColl-Tyrosine-RGDS, 90% HyColl and 10% HyColl-Tyrosine-RGDS, 75% HyColl and 25% HyColl-Tyrosine-RGDS, 50% HyColl and 50% HyColl-Tyrosine-RGDS, 0% HyColl and100% HyColl-Tyrosine-RGDS.
  • Proliferation Assay which directly measures the number of the metabolically active cells in the culture by the cleavage of tetrazolium salts to formazan. After culturing C2C12 cells on protein coated PS plates for 24 hours, 20 ⁇ _ of WST-1 reagent was added into each well and incubated for 3 h. The absorbance at 440 nm was then monitored with a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
  • C2C12 cells were seeded onto the hydrogels and subjected to a live/dead assay including Calcein AM (green-stain for live cells) and Ethidium homodimer-1 (red-stain for dead cells). Prior to culturing, the hydrogels were sterilized with antibiotic solution (penicillin-streptomycin), followed by thorough washing with PBS. The C2C12 cells with a density of approximately 200 cells/mm 2 were cultured on the sterilized Hycoll-Tyrosine-RGDS hydrogels for 24 hours.
  • antibiotic solution penicillin-streptomycin
  • the cell culture medium was then replaced with DPBS containing 2uM Calcein AM and 4uM Ethidium homodimer-1 and incubated for 45 minutes.
  • the hydrogels were then washed 3 times with DPBS.
  • the cells were observed using a confocal fluorescent microscope (Zeiss, Germany).
  • Example 1 A Structurally Stable, Highly Pure HyColl, HyColl-RGD Product can be
  • CL has a propensity to form both fibrils and aggregates at neutral pH. Increasing the length of the CL construct has been shown to increase both fibril diameter and fibril length (International Patent Application Publication No. WO 2010/091251). It has been stated that the addition of poly ethylene glycol compounds (PEG) may be required to enhance the solubility of collagen-like proteins (International Patent Application Publication No. WO 2015/031950).
  • PEG poly ethylene glycol compounds
  • HyColl and HyColl-RGD were found to be highly soluble (> 300 mg/ml) in ddH 2 0 as well as in acidic, neutral and basic buffers such as citric acid/Na 2 HP0 4 buffer (pH 4), citric acid/Na 2 HP0 4 buffer (pH 7), Na 2 HP04/NaH 2 P04 buffer (pH 7),
  • HyColl NaHC0 3 /Na 2 C0 3 buffer (pH 10), and PBS (pH 7.4) at room temperature.
  • HyColl was also found to be soluble in MES buffer (pH 4.5). In all buffers, no precipitation was observed.
  • HyColl and HyColl-RGD show a single band demonstrating that we are able to manufacture and purify both HyColl and HyColl-RGD.
  • the single band indicates that no degradation has occurred and that we can achieve high purity.
  • LC-MS Liquid Chromatography-Mass Spectrometry
  • the band at 1654 cnr 1 is characteristic of alpha-helical structure associated with the V domain.
  • Example 2 The CD Wavelength Spectra for HyColl in Acidic, Physiologic and Basic Buffers Resemble that of Triple-Helical Collagen with an Ellipticity Maximum at 220 nm and a Minimum at 198 nm.
  • CD wavelength scans were carried out for 10 ⁇ HyColl and HyColl-RGD solutions respectively in citric acid/Na 2 HP0 4 buffer (pH 4), citric acid/Na 2 HP0 4 buffer (pH 7), Na 2 HP0 4 /NaH 2 P0 4 buffer (pH 7) and NaHC0 3 /Na 2 C0 3 buffer (pH 10). Additional CD scans were carried out in the buffers, MES (pH 4.5) and PBS (pH 7.4) that were used to prepare hydrogels and a HyColl-based bio-ink for 3D bio-printing.
  • Figures 7 and 8 show the plots of the mean residue molar ellipticity [ ⁇ ] of HyColl and HyColl-RGD respectively at 20°C as a function of wavelength ⁇ in buffers with different pH.
  • the spectra show the characteristic features of a collagen or collagen-like triple helix with a maximum peak at 220 nm and a minimum at 198 nm for all buffers.
  • V and BBB domains contribute additively to the observed CD spectra. It has been previously shown that the recombinant V domain consists of 25.7% ( ⁇ 4.3%) a- helices, 43.6 ( ⁇ 10.5%) ⁇ -sheets, and 30.7% ( ⁇ 1 1.3%) other secondary structure elements (1).
  • Example 3 The Thermal Stability of HyColl is pH Dependent and Comparable to Human Collagen at Physiologic pH.
  • HyColl Thermally-induced denaturation of the protein was examined by monitoring the mean residue molar ellipticity [ ⁇ ] as a function of increasing temperature at 220 nm. Plots for HyColl are provided in Figures 9 to 13).
  • HyColl is heated to 50°C, the protein denatures and takes on a random coil structure as expected.
  • the temperature at which denaturation occurs for HyColl in the acidic, basic and neutral pH buffers occurs within a narrow temperature range ( ⁇ ⁇ 4°C) as shown in Table 3.
  • the thermal stability of HyColl is comparable to that of human collagen which has been shown by CD to denature above 37°C (Bentz, H., Bachinger, H.P., Glanville, R., and Kuhn, K. (1978) Eur, J.
  • HyColl The denaturation temperature was found to be pH dependent; lowest at acidic and basic pH and highest at neutral pH. This data indicates that the thermal stability of HyColl may be affected by the percentage of charged residues in the protein suggesting that at neutral pH electrostatic interactions play a role in HyColl stability.
  • Table 3 The thermal stability for HyColl at acidic, neutral and basic pH as determined by CD spectroscopy.
  • Example 4 Insertion of Cell Binding Sequences such as RGD Can Be Made Without Negatively Affecting the Thermal Stability of the Protein.
  • HyColl-RGD Plots are also provided for HyColl-RGD in Figures 14 to 18.
  • the temperature at which denaturation occurs for HyColl-RGD in the acidic, basic and neutral pH buffers occurs within the same narrow temperature range ( ⁇ ⁇ 4°C) as shown Table 4.
  • This data demonstrates that short cell binding integrin ligands such as RGD can be inserted into the HyColl sequence without negatively affecting the thermal stability of the protein.
  • the thermal stability of HyColl-RGD at physiologic pH remains comparable to that of human collagen.
  • Example 5 Thermal Denaturation of HyColl is a pH Dependent, Reversible Process.
  • Example 6 Similar Thermal Transitions are Seen by Micro-Differential Scanning Calorimetry (Micro-DSC).
  • Denaturation temperatures (Tm) in the range of 35.2°C to 37.4°C for 100 ⁇ HyColl were observed for acidic, basic and physiologic pH, comparable to that observed by CD.
  • the cooling scans show that refolding of HyColl occurs between 26°C and 32°C in different pH buffers, indicating that refolding requires time to equilibrate, which has been previously demonstrated by CD. Secondary peaks observed at pH 4 and pH 10 may be due to soluble aggregates that are reversible with time. 10 ⁇ of HyColl shows the similar thermal scans with 100 ⁇ of HyColl with denaturation temperatures observed in the 34.7°C to 37.3°C range. The higher Tm observed with DSC compared to that observed by CD is due to faster heating conditions and non-equilibrium conditions.
  • the cooling scans show that refolding of HyColl occurs between 26°C and 32°C in pH 4 and pH 7 buffers, indicating that refolding requires time to equilibrate.
  • the protein does not refold in MES buffer (pH 4.5).
  • pH 10 the spectrum shows a tiny transition peak in the second heating scan which indicates a very slow recovery rate or a non-reversible thermal transition.
  • the thermal stability data given in Table 6 for HyColl-RGD indicates that the cell binding sequence RGD can be inserted into the HyColl without negatively affecting the thermal stability of the protein.
  • Example 7 HyColl and HyColl-RGD Can Be Chemically Crosslinked to Produce Thermally Stable Transparent Hydrogels.
  • HyColl and HyColl-RGD hydrogels were denatured.
  • the thermal stability of the fabricated hydrogels is given in Table 7.
  • the denaturation temperature for HyColl increases from 37.27°C to 46.40°C upon the addition of EDC/NHS in MES (pH 4.5) and increases from 37.58°C to 62.50°C upon the addition of DMTMM in PBS (pH 7.4) demonstrating that HyColl and HyColl-RGD can be covalently crosslinked to produce thermally stable hydrogels.
  • Example 8 Mechanical Properties Equivalent to Collagen Hydrogels Can Be Achieved.
  • HyColl hydrogels prepared from 10% (w/w) solutions and crosslinked with EDC/NHS in MES (pH 4.5) buffer are shown in Table 8.
  • Table 8 For mechanical property comparisons, we also prepared hydrogels from porcine derived type I atello-collagen using the same concentrations and a crosslinking ratio of 0.5.
  • the tensile strength, modulus and elongation at break for porcine collagen hydrogel was 0.7395 ⁇ 0.0433, 4.31 1 ⁇ 0.192 and 29.55 ⁇ 2.91 MPa respectively, demonstrating that equivalent mechanical properties of HyColl hydrogel material can be achieved.
  • the collagen solution pH was approximately 2 and required pH adjustment using sodium hydroxide to enable EDC/NHS crosslinking.
  • no pH adjustment was required.
  • the storage and loss moduli are nearly independent of frequency, with G ' (u)»G " (u), indicative of a nearly ideal viscoelastic solid.
  • the overlap of the data before and after application of the non-linear deformation indicates that the gels are fully reversible in their response to large strains, another highly desirable trait for application of these materials.
  • Example 9 HyColl Hydrogels have Improved Resistance to Collagenase Compared to Collagen Hydrogels.
  • Example 1 1 A series of Variants were Constructed and Showed the Similar CD Wavelength Spectra, Solubility and Thermal Stability in Acidic, Physiologic and Basic pH with HyColl.
  • cleavage tags such as DDDK, IEGR, ENLYFQG, and LVPRGS were also designed to be inserted into the sequence between the V domain and the B domain.
  • Protein purity of HyColl-Tyrosine- RGDS was confirmed with SDS-PAGE, as shown in Figure 46. The single band indicates that high purity was achieved and that no degradation of the product occurred.
  • CD wavelength scans were carried out for 10 ⁇ HyColl-Tyrosine-RGDS solutions respectively in citric acid/Na 2 HP0 4 buffer (pH 4), Na 2 HP04/NaH 2 P04 buffer (pH 7), PBS (pH7.4) and NaHC03/Na2C03 buffer (pH 10).
  • Figure 47 shows the plots of the mean residue molar ellipticity [ ⁇ ] of HyColl -Tyrosine-RGDS at 20°C as a function of wavelength ( ⁇ ) in buffers with different pH.
  • the spectra show the characteristic features of a collagen or collagen-like triple helix with a maximum peak at 220 nm and a minimum at 198 nm for all buffers.
  • HyColl-Tyrosine-RGDS Plots are provided for HyColl-Tyrosine-RGDS in Figures 48 to 51.
  • the temperature at which denaturation occurs for HyColl-Tyrosine-RGDS in the acidic, basic and neutral pH buffers occurs within the same narrow temperature range as shown Table 9.
  • Na2HPC /NaH2P04 (pH 7) is readily reversible.
  • This data demonstrates that mutation of amino acids (from threonine to tyrosine), insertion of cell-binding domain such as RGDS and insertion of linker sequences between the B domain and cell-binding domain such as GSTSGSGT into the HyColl sequence do not affecting the thermal stability of the protein.
  • Table 9 The thermal stability for HyColl-Tyrosine-RGDS at acidic, neutral and basic pH as determined by CD spectroscopy.
  • RGDS:HyColl On the surfaces coated with 100% HyColl, C2C12 cells remained rounded and unattached after 24 hours of culture. In contrast, C2C12 cells attached and spread on the surfaces coated with as little as 5% HyColl-Tyrosine-RGDS. Cell attachment and spreading was improved by increasing the ratio of HyColl-Tyrosine-RGDS. The metabolic activity of the C2C12 cells was also investigated.
  • Figure 58 shows the relative metabolic activity of C2C12 cells after culturing for 24 hours as a function of the ratio of HyColl- Tyrosine-RGDS:HyColl coated onto the surfaces.
  • C2C12 cells increased as the ratio of HyColl-Tyrosine-RGDS on the surface was increased, reaching 75% of maximal metabolic activity for surface coatings with approximately 10% RGDS ligand demonstrating that HyColl-Tyrosine-RGDS improves the attachment and viability of cells by increasing the amount of available cell binding ligand.
  • HyColl-Tyrosine-RGDS can also be crosslinked using SPS only by exposing the material to white light. This crosslinking reaction took more time to occur compared to those gels made using a 455 nm light source but over time produces a transparent hydrogel material as shown in Figure 61 .
  • HyColl-Tyrosine-RGDS solution was used to determine the temperature at which the photo-crosslinked HyColl- Tyrosine-RGDS hydrogels were denatured.
  • the thermal stability of the fabricated hydrogels is given in Table 1 1 .
  • the denaturation temperature for HyColl-Tyrosine-RGDS solution increased from 36.64°C to 70.39°C upon photo-crosslinking in the presence of Ru(bpy) 3 CI 2 and SPS.
  • HyColl-Tyrosine-RGDS is a 10% (w/v) solution prepared in 0.1 M PBS.
  • Example 14 HyColl-Tyrosine-RGDS Can Be Enzyme Crosslinked to Produce Thermally Stable Transparent Hydrogels
  • HyColl-Tyrosine-RGDS hydrogels were denatured.
  • the thermal stability of the fabricated hydrogels is given in Table 12.
  • the denaturation temperature for HyColl-Tyrosine-RGDS increased from 36.64°C to 51 .04 °C upon mixing with 24U HRP and slightly increased further to 53.00 °C upon mixing with 48U HRP.
  • Figure 63 shows the emission spectrum with excitation at 260 nm demonstrating that as the units HRP is added to the protein solution the number of available tyrosines for crosslinking decreases as shown by the decreasing intensity of the 305 nm peak representative of tyrosine.
  • Table 12 Thermal Stabilit of Enz me Crosslinked H Coll-T rosine-RGDS H dro els
  • Example 15 Bio-Functionality of Photo and Enzyme Cross-linked Hydrogels.
  • HyColl-Tyrosine-RGDS hydrogels are bio-functional and biocompatible.
  • the foregoing description and examples have been set forth merely to illustrate the invention and are not intended as being limiting. Each of the disclosed aspects and embodiments of the present invention may be considered individually or in combination with other aspects, embodiments, and variations of the invention. In addition, unless otherwise specified, none of the steps of the methods of the present invention are confined to any particular order of performance.

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Abstract

L'invention concerne un polypeptide codant pour une protéine de type collagène modifiée ou un fragment de celle-ci, la protéine de type collagène modifiée comprenant au moins l'une des caractéristiques suivantes: est plus soluble dans des solutions acides, neutres et/ou basiques qu'une protéine de type collagène non modifiée ou un fragment de celle-ci; est stable en solution d'environ 5 à environ 36 °C à un pH physiologique; s'auto-assemble en une triple hélice dans des solutions acides, neutres et/ou basiques; a une stabilité thermique qui est environ la même ou meilleure que le collagène; lorsqu'elle est réticulée dans un hydrogel, est stable à au moins 37 °C; et est plus résistante à la collagénase que le collagène.
PCT/CA2018/051077 2017-09-06 2018-09-06 Protéines de type collagène Ceased WO2019046943A1 (fr)

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US11028148B2 (en) 2017-09-28 2021-06-08 Geltor, Inc. Recombinant collagen and elastin molecules and uses thereof
CN113025599A (zh) * 2021-04-02 2021-06-25 重庆科润生物医药研发有限公司 一种重组溶组织梭菌i型胶原酶及其制备方法和应用
US11168126B2 (en) 2019-04-12 2021-11-09 Geltor, Inc. Recombinant elastin and production thereof
US11174300B2 (en) 2020-01-24 2021-11-16 Geltor, Inc. Animal-free dietary collagen
WO2023016895A1 (fr) * 2021-08-09 2023-02-16 Evonik Operations Gmbh Polynucléotide codant pour une protéine bactérienne de type collagène
WO2023041689A1 (fr) * 2021-09-20 2023-03-23 Evonik Operations Gmbh Hydrogels de type collagène non adhésifs
CN116407682A (zh) * 2022-11-09 2023-07-11 内蒙古和讯生物科技有限公司 一种基因编码的重组类胶原蛋白超分子水凝胶制备方法和应用
WO2024002806A1 (fr) * 2022-07-01 2024-01-04 Evonik Operations Gmbh Protéines de type collagène bactériennes recombinantes photoréticulables
WO2024165412A1 (fr) * 2023-02-09 2024-08-15 Evonik Operations Gmbh Composition cosmétique comprenant une protéine de type collagène (clp) bactérienne recombinante et ses utilisations
CN118530476A (zh) * 2024-04-19 2024-08-23 美尔健（深圳）生物科技有限公司 一种ii型重组人胶原蛋白水凝胶的制备方法及其应用
WO2024188850A1 (fr) * 2023-03-16 2024-09-19 Evonik Operations Gmbh Protéines de type collagène bactériennes recombinantes thiolées

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WO2018152444A1 (fr) * 2017-02-17 2018-08-23 The Texas A&M University System Agents thérapeutiques à base de collagène procaryote pour adhésions post-opératoires

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US11180541B2 (en) 2017-09-28 2021-11-23 Geltor, Inc. Recombinant collagen and elastin molecules and uses thereof
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US11168126B2 (en) 2019-04-12 2021-11-09 Geltor, Inc. Recombinant elastin and production thereof
US12269865B2 (en) 2019-04-12 2025-04-08 Geltor, Inc. Recombinant elastin and production thereof
US11174300B2 (en) 2020-01-24 2021-11-16 Geltor, Inc. Animal-free dietary collagen
US11332505B2 (en) 2020-01-24 2022-05-17 Geltor, Inc. Animal-free dietary collagen
CN113025599B (zh) * 2021-04-02 2023-09-12 重庆科润生物医药研发有限公司 一种重组溶组织梭菌i型胶原酶及其制备方法和应用
CN113025599A (zh) * 2021-04-02 2021-06-25 重庆科润生物医药研发有限公司 一种重组溶组织梭菌i型胶原酶及其制备方法和应用
WO2023016895A1 (fr) * 2021-08-09 2023-02-16 Evonik Operations Gmbh Polynucléotide codant pour une protéine bactérienne de type collagène
WO2023041689A1 (fr) * 2021-09-20 2023-03-23 Evonik Operations Gmbh Hydrogels de type collagène non adhésifs
WO2024002806A1 (fr) * 2022-07-01 2024-01-04 Evonik Operations Gmbh Protéines de type collagène bactériennes recombinantes photoréticulables
CN116407682A (zh) * 2022-11-09 2023-07-11 内蒙古和讯生物科技有限公司 一种基因编码的重组类胶原蛋白超分子水凝胶制备方法和应用
WO2024165412A1 (fr) * 2023-02-09 2024-08-15 Evonik Operations Gmbh Composition cosmétique comprenant une protéine de type collagène (clp) bactérienne recombinante et ses utilisations
WO2024188850A1 (fr) * 2023-03-16 2024-09-19 Evonik Operations Gmbh Protéines de type collagène bactériennes recombinantes thiolées
CN118530476A (zh) * 2024-04-19 2024-08-23 美尔健（深圳）生物科技有限公司 一种ii型重组人胶原蛋白水凝胶的制备方法及其应用

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