COLLAGEN-LIKE PROTEINS

Field

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, products, and methods.

Background

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. The defining feature of collagen is its molecular structure, including a unique super coiled triple-helix. Although collagens were initially thought to be found only in multi-cellular animals, it has been recently shown that there are collagen-like proteins in bacteria that adopt a triple-helix structure, with a thermal stability similar to that seen for human collagen. For example, 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

Lukomski, S. (2002) J. Biol. Chem. 277, 27312-27318; Yu, Z, Brodsky, B., and Inouye, M. (201 1) J. Biol. Chem 286, 18960-18968).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.

Recombinant V-CL has been shown to be only partially soluble at pH 7 and to precipitate as poorly ordered aggregates, thus limiting its use for biomedical and regenerative medicine applications. Thus, there have been limited reports about the fabrication of bacterial collagen and collagen-like proteins with properties suitable for medical applications (Yu, Z., An, B., Ramshaw, J., and Brodsky, B. (2014) Journal of Structural Biology 186,451-461). For example, International Patent Application Publication No. WO 2015/031950 describes recombinant or synthetic collagen-like proteins comprising at least one triple-helical domain and wherein the collagen-like protein is modified compared to a native bacterial collagen-like sequence. However, use of these materials has been primarily limited due to poor solubility and propensity to form fibrils at neutral pH (Yoshizumi, A., Yu, Z., Silva, T., Thiagarajan, G., Ramshaw, J., Inouye, M. and Brodsky, B. (2009) Protein Science 18, 1241-1251). Thus the majority of applications involve the manufacture of sponge-like structures that require the stabilization of freeze-dried material with chemical crosslinkers such as glutaraldehyde (Peng, Y., Yoshizumi, A., Danon, S., Glattauer, V., et al. Biomaterials (2010) 31 , 2755-2761), foam- based applications that require solubilization of the protein in acetic acid followed by a dehydrothermal crosslinking procedure (Parmar, P., St. Pierre, J., Chow, L, Puetzer, J. et al. Advanced Health Care Materials (2016) 5(13), 1656 - 1666) and conjugation of the Scl2 protein to a supporting hydrogel network (Cosgriff-Hernadez, E. , Hahn, M.S., Russel, B., Wilems, T., Munoz-Pinto, D. , et al. (2010) Acta Biomater. 6, 3969-3977; Browning, M.B., Dempsey, D., Guiza, V., Becerra, S. et al. (2012) Acta Biomater. 8, 1010-1021). Thus there remains a need for novel recombinant collagen or collagen-like products with different and/or improved attributes.

Summary

In accordance with an aspect, there is provided a polypeptide comprising a sequence with at least 70% sequence identity to the sequence:

MNHKVHMHHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLL T YLQEREQAENSWRKRLLKGIQDHALDGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGE

QGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGKDGERGPVGPAGPKGEQGPQGL

PGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGK

DGERGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGD

RGETGPKGPKGERG E AG PAG KD G E RG P VG PA (SEQ ID NO: 1)

or a fragment thereof.

In an aspect, the polypeptide comprises a sequence with at least 75%, 80%, 85%,

90%, 95%, or 99% identity to the sequence of SEQ ID NO: 1 or a fragment thereof.

In an aspect, the polypeptide comprises the sequence of SEQ ID NO: 1 or a fragment thereof.

In an aspect, the polypeptide comprises the sequence of SEQ ID NO: 1.

In an aspect, the sequence

MNHKVHMHHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLT YLQEREQAENSWRKRLLKGIQDHALD or a fragment or variant thereof is cleaved from the polypeptide after expression.

In an aspect, the polypeptide further comprises a bioactive domain.

In an aspect, 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.

In an aspect, the cell binding domain comprises at least one of RGD, RGDS, DGEA, REDV, YIGSR, IKVAV, KAFAK, and VAPG. In an aspect, the biomineralization domain comprises at least one of MLPHHGA, SVSVGMKPSPRP, ESQES, and QESQSEQDS.

In an aspect, the metallization domain comprises at least one of NPSSLFRYLPSD, MHGKTQATSGTIQS, AQNPSDNNTHTH, and RLELAIPLQGSG.

In an aspect, the elastomeric domain comprises at least one of VPGVG, (AG) ₃ EG,

GYSGGRPGGQDLG, GGFGGMGGGS, PGQGQQ, and GYYPTSPQ.

In an aspect, the bioactive domain is linked to the C-terminal end of the polypeptide.

In an aspect, the bioactive domain is linked to the polypeptide with a linker sequence.

In an aspect, the linker sequence comprises (GSTSGSGT) _n or

(GSTSGSGKPGSGEGSTKGT)n, wherein n is an integer.

In an aspect, the polypeptide is modified to include cross-linking elements.

In an aspect, the cross-linking elements are primary amines, aromatics, and/or glutamine.

In an aspect, the cross-linking elements are selected from lysine, tyrosine, tryptophan, phenylalanine, glutamine, histidine and combinations thereof.

In an aspect, the polypeptide further comprises one or more terminal cysteines for conjugation to other polymers and/or peptides.

In an aspect, the polypeptide is ionically crosslinked.

In an aspect, the polypeptide is ionically crosslinked with cationic and/or anionic crosslinkers, such as tri-poly-phosphate or spermidine.

In accordance with an aspect, there is provided a polypeptide comprising the sequence

GPXGEQGPQGLPGKDGEAGAQGPAGPMGPAGEXGEKGEPGTXGAKGDRGETGPXGPX 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.

In accordance with an aspect, there is provided a 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.

In accordance with an aspect, there is provided a 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.

In an aspect, the polypeptide is repeated consecutively 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

In an aspect, the polypeptide further comprises a V domain of a collagen-like protein, wherein the V domain is optionally cleaved after expression of the polypeptide.

In accordance with an aspect, there is provided a polypeptide encoding a modified collagen-like protein or fragment thereof, wherein the modified collagen-like protein comprises at least one of the following features:

is more soluble in acidic, neutral, and/or basic solutions than a non-modified collagen like protein or fragment thereof;

is stable in solution at from about 5 to about 36°C at physiologic pH;

self-assembles into a triple helix in acidic, neutral, and/or basic solutions;

has thermal stability that is about the same or better than collagen;

when crosslinked into a hydrogel is stable to at least 37°C; and

is more collagenase-resistant than collagen.

In accordance with an aspect, there is provided 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.

In an aspect, the modified collagen-like protein is modified to increase availability of lysine, tyrosine, tryptophan, phenylalanine, glutamine, histidine or combinations thereof.

In accordance with an aspect, there is provided a collagen-like protein comprising the polypeptide described herein.

In an aspect, the collagen-like protein is of bacterial origin and is modified to comprise the polypeptide described herein.

In an aspect, the collagen-like protein is derived from Streptococcus pyogenes. In an aspect, the collagen-like protein is a modified Scl2 protein.

In an aspect, a naturally occurring portion of the collagen-like protein is substituted with the polypeptide described herein.

In an aspect, 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.

In an aspect, the CL domain consists of at least one copy of the polypeptide described herein. In an aspect, the polypeptide described herein is present in one or more substantially consecutive repeats.

In accordance with an aspect, there is provided a collagen-like CL domain of a collagen-like protein comprising the polypeptide described herein.

In an aspect, the CL domain is of bacterial origin and is modified to comprise the polypeptide described herein.

In an aspect, the CL domain is derived from Streptococcus pyogenes.

In an aspect, the CL domain is a modified Scl2 CL domain.

In an aspect, a naturally occurring portion of the CL domain is substituted with the polypeptide described herein.

In an aspect, 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.

In an aspect, the CL domain consists of at least one copy of the polypeptide described herein.

In an aspect, the polypeptide described herein is present in one or more substantially consecutive repeats.

In accordance with an aspect, there is provided a nucleic acid molecule encoding the polypeptide, the collagen-like protein, or the CL domain described herein.

In accordance with an aspect, there is provided an expression vector comprising the nucleic acid molecule described herein.

In accordance with an aspect, there is provided a recombinant host cell comprising the expression vector described herein.

In accordance with an aspect, there is provided a recombinant host cell expressing, displaying, and/or secreting the polypeptide, the collagen-like protein, or the CL domain described herein.

In accordance with an aspect, there is provided a composition comprising the polypeptide, the collagen-like protein, or the CL domain described herein.

In an aspect, the composition further comprises a pharmaceutically acceptable carrier, diluent, and/or buffer.

In an aspect, the composition is transparent.

In an aspect, the composition is a hydrogel.

In an aspect, the hydrogel is produced by chemical, physical, ionic, photo, or enzyme based crosslinking methods.

In an aspect, the hydrogel is thermally stable at physiologic temperature (37°C). In an aspect, the composition further comprises at least one of cells, growth factors, nanoparticles, other proteins and polymers. In accordance with an aspect, there is provided a bio-ink comprising the composition described herein.

In accordance with an aspect, there is provided an ophthalmic device comprising the composition described herein.

In accordance with an aspect, there is provided a dermal implant comprising the composition described herein.

Brief Description of the Figures

The present invention will be further understood from the following description with reference to the Figures, in which:

Figure 1. The nucleic acid and polypeptide sequence of the HyColl Sequence.

Figure 2. Cell binding designs: (A) HyColl-RGD Sequence; (B) HyColl-S-RGD with a GSTSGSGT (spacer, S).

Figure 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.

Figure 3B. A generic biofunctional HyColl design showing modifications to include crosslinking elements.

Figure 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 protein™ standards; Lane 2: Purified HyColl; Lane 3: Purified HyColl-RGD.

Figure 5. An LC-MS spectra for HyColl. A molecular weight of 32875.64 Da was calculated using adjacent charge states.

Figure 6. An FTIR spectra for HyColl.

Figure 7. CD wavelength scans of 10 μΜ HyColl 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.

Figure 8. CD wavelength scans at 10 μΜ 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.

Figure 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 1. CD thermal scans of 10 μΜ 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).

Figure 12. CD thermal scans of 10 μΜ HyColl in MES (pH 4.5) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).

Figure 13. CD thermal scans of 10 μΜ HyColl 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).

Figure 14. CD thermal scans of 10 μΜ 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 15. CD Thermal scans of 10 μΜ HyColl-RGD in citric acid and Na ₂ HP0 (pH 7) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).

Figure 16. CD thermal scans of 10 μΜ HyColl-RGD in NaH ₂ P0 and Na ₂ HP0 (pH 7) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).

Figure 17. CD thermal scans of 10 μΜ HyColl-RGD in PBS buffer (pH 7.4) showing the denaturation with increasing temperature (squares) and refolding of the protein with decreasing temperature (triangles).

Figure 18. CD thermal scans of 10 μΜ 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).

Figure 19. CD wavelength scans of 10 μΜ 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.

Figure 20. Micro-DSC heating and cooling scan for 100 μΜ HyColl in Na ₂ HP0 and citric acid (pH 4).

Figure 21. Micro-DSC heating and cooling scan for 100 μΜ HyColl in MES (pH 4.5). Figure 22. Micro-DSC heating and cooling scan for 100 μΜ HyColl in Na ₂ HP0 and citric acid (pH 7). Figure 23. Micro-DSC heating and cooling scan for 100 μΜ HyColl in Na ₂ HP0 ₄ and NaH ₂ P0 ₄ (pH 7).

Figure 24. Micro-DSC heating and cooling scan for 100 μΜ HyColl in Na ₂ C0 ₃ and NaHC0 ₃ (pH 10).

Figure 25. Micro-DSC heating and cooling scan for 10 μΜ HyColl in Na ₂ HP0 ₄ and citric acid (pH 4).

Figure 26. Micro-DSC heating and cooling scan for 10 μΜ HyColl in MES (pH 4.5). Figure 27. Micro-DSC heating and cooling scan for 10 μΜ HyColl in Na ₂ HP0 ₄ and citric acid (pH 7).

Figure 28. Micro-DSC heating and cooling scan for 10 μΜ HyColl in Na ₂ HP0 and

NaH ₂ P0 ₄ (pH 7)

Figure 29. Micro-DSC heating and cooling scan for 10 μΜ HyColl in Na ₂ C0 ₃ and NaHC0 ₃ (pH 10).

Figure 30. Micro-DSC heating and cooling scan for 100 μΜ HyColl-RGD in Na ₂ HP0 ₄ and citric acid (pH 4).

Figure 31. Micro-DSC heating and cooling scan for 100 μΜ HyColl-RGD in Na ₂ HP0 and citric acid (pH 7).

Figure 32. Micro-DSC heating and cooling scan for 100 μΜ HyColl-RGD in PBS (pH

7.4).

Figure 33. Micro-DSC heating and cooling scan for 100 μΜ HyColl-RGD Na ₂ HP0 ₄ and NaH ₂ P0 ₄ (pH 7).

Figure 34. Micro-DSC heating and cooling scan for 100 μΜ HyColl-RGD in Na ₂ C03 and NaHC0 ₃ (pH 10).

Figure 35. Micro-DSC heating and cooling scan for 10 μΜ HyColl-RGD in Na ₂ HP0 and citric acid (pH 4).

Figure 36. Micro-DSC heating and cooling scan for 10 μΜ HyColl-RGD in Na ₂ HP0 and citric acid (pH 7).

Figure 37. Micro-DSC heating and cooling scan for 10 μΜ HyColl-RGD in PBS (pH

7.4).

Figure 38. Micro-DSC heating and cooling scan for 10 μΜ HyColl-RGD in Na ₂ HP0 and NaH ₂ P0 ₄ (pH 7).

Figure 39. Micro-DSC heating and cooling scan for 10 μΜ HyColl-RGD in Na ₂ C0 ₃ and NaHCOs (pH 10).

Figure 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. Figure 41 . Dynamic moduli, G ^' (solid squares) and G ^" (open squares) of a 5% wt. HyColl gel as a function of strain amplitude at fixed frequency, ω = 1 s ¹ . The reduction of G ^' (YO) and the concomitant rise of G ^" (yo) beyond (γ ₀ = 10%) is a sign of non-linear strain softening of the gel.

Figure 42. Dynamic moduli, G ^' (solid symbols) and G ^" (open symbols) of a 5% (w/w)

HyColl hydrogel as a function of frequency (ω) at fixed strain amplitude (γ ₀ = 0.1 %). The solid and open squares show data measured on a previously unperturbed sample, while the solid and open triangles show data obtained after the application of a large shear amplitude deformation (γ ₀ =100%).

Figure 43. Comparison of HyColl to porcine collagen in vitro collagenase

degradation.

Figure 44. Scanning electron microscopy (Cryo-SEM) image of a section of an EDC/NHS crosslinked HyColl hydrogel (Scale = 500μηι).

Figure 45A. Amino acid sequence of the HyColl-DGEA

Figure 45B. Amino acid sequence of the HyColl- MLPHHGA

Figure 45C. Amino acid sequence of the HyColl-Tyrosine-RGDS

Figure 45D. Amino acid sequence of the HyColl-Tyrosine-RGDS-V cut

Figure 46. A Gelcode blue stained SDS-PAGE gel showing the purification of HyColl- Tyrosine-RGDS. The lanes contain the following: Lane 1 : Precision plus protein™ standards; Lane 2: Purified HyColl-Tyrosine-RGDS.

Figure 47. CD wavelength scans of 10 μΜ 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.

Figure 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).

Figure 49. CD thermal scans of 10 μΜ 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 μΜ HyColl-Tyrosine-RGDS in PBS (pH7.4) showing the denaturation with increasing temperature (triangles) and refolding of the protein with decreasing temperature (dots).

Figure 51 . CD thermal scans of 10 μΜ 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 μΜ HyColl-Tyrosine-RGDS in Na ₂ HP0 ₄ and citric acid (pH 4).

Figure 53. Micro-DSC heating and cooling scan for 100 μΜ HyColl-Tyrosine-RGDS in Na ₂ HP0 ₄ and NaH ₂ P0 ₄ (pH 7).

Figure 54. Micro-DSC heating and cooling scan for 100 μΜ HyColl-Tyrosine-RGDS in

PBS (pH 7.4).

Figure 55. Micro-DSC heating and cooling scan for 100 μΜ HyColl-Tyrosine-RGDS in Na ₂ C0 ₃ and NaHC0 ₃ (pH 10).

Figure 56. C2C12 cells on 25uM Hycoll(a) and Hycoll-RGD(b) solution coated PS surface.

Figure 57. C2C12 cells on different percentage of HyColl-Tyrosine-RGDS coated PS surfaces.

Figure 58. WST-1 assay on 25uM PS surface coated with different percentage of HyColl-Tyrosine-RGDS.

Figure 59. HyColl-Tyrosine-RGDS Photo-crosslinked hydrogel using SPS only (blue light exposure).

Figure 60. Fluorescence Spectrum for HyColl-Tyrosine-RGDS Photo-crosslinked hydrogel using SPS only (blue light exposure). Excitation at 315 nm.

Figure 61 . HyColl-Tyrosine-RGDS Photo-crosslinked hydrogel using SPS only (white light exposure).

Figure 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.

Detailed Description

In order to overcome the limited use of V-CL, 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. 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 , designated herein as HyColl (SEQ ID NO: 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:

Carbon (C) 1354

Hydrogen (H) 2154

Nitrogen (N) 434

Oxygen (O) 464

Sulfur (S) 6

Therefore, described herein is a recombinant collagen-like polypeptide that is highly soluble in aqueous solutions at physiologic pH as well as in both acidic and basic buffers (i.e. at pHs above and below the estimated pi). 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. This high solubility, particularly at physiologic pH (pH 7.4), provides a distinct advantage over collagen and V-CL as this allows the material to be used, for example, to fabricate hydrogels or bio-inks for 3D bio-printing applications. Bio-ink solutions containing the recombinant collagen-like polypeptide described herein can be made at elevated concentrations, either alone or in combination with a variety of cell types without the risk of protein precipitation occurring during the printing process and subsequent stabilization of the printed three dimensional construct.

Further demonstrated herein is that 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. Furthermore, 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). And unlike animal-derived collagen, the sequence can be modified to enhance or change specific biological or functional properties. As previously stated, modification of the native amino acid sequence to increase the availability of key functional groups (e.g. primary amines, aromatics, and/or glutamine) allows one skilled in the art to use numerous crosslinking approaches to stabilize the protein (e.g. chemical and photo-crosslinking). This, along with its high solubility across a broad pH range, makes the material described herein particularly well-suited to bio-printing. Definitions

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287- 9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the typical materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

In understanding the scope of the present application, the articles "a", "an", "the", and "said" are intended to mean that there are one or more of the elements.

Additionally, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

It will be understood that any aspects described as "comprising" certain components may also "consist of" or "consist essentially of," wherein "consisting of has a closed-ended or restrictive meaning and "consisting essentially of" means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase "consisting essentially of"

encompasses any known pharmaceutically acceptable additive, excipient, diluent, carrier, and the like. Typically, a 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.

It will be understood that any component defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation.

Terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms may refer to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 20% or ± 10%, more typically ± 5%, even more typically ± 1 %, and still more typically ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." The word "or" is intended to include "and" unless the context clearly indicates otherwise.

Throughout this disclosure, various aspects described herein can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope described herein. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Many patent applications, patents, and publications are referred to herein to assist in understanding the aspects described. Each of these references is incorporated herein by reference in their entirety.

Unless otherwise specified, a "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).

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. As used herein, 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.

The terms "therapeutically effective amount", "effective amount" or "sufficient amount" mean 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.

Moreover, 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. It will also be appreciated that 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.

The term "subject" as used herein refers to any member of the animal kingdom, typically a mammal. The term "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.

The term "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.

The term "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.

"Active" or "activity" for the purposes herein 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.

Thus, "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. Ordinarily, however, 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

physiological, chemical, or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered "substantially identical" polypeptides. Conservative amino acid mutation 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).

In a non-limiting example, 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. By the term "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). By the term "neutral amino acid" (also "polar amino acid"), it is meant 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). The term "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. For example, and without wishing to be limiting, 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. 7,981 ,632, 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.

In another example, 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. As is also known to those of skill in the art, linker sequences may be used in conjunction with the additional sequences or tags. Additionally, 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.

Any suitable expression vector can be used. For example, 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 2μ 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. Biol, 159:601-664 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Nat'l Acad. Sci USA, 80:4654-4659 (1983); Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, (1980), all of which are incorporated by reference herein).

In aspects, 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. Examples of 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. Also described herein are recombinant host cells containing the expression vectors previously described. Nucleic acids, which comprise a sequence encoding a polypeptide according to the invention, can be used for transformation of a suitable mammalian host cell.

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.

These 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. Acids Res., 14:4683-4690 (1986), all of which are incorporated by reference herein) at the 5' end of the antibody-encoding gene of interest. These secretory leader peptide elements can be derived from either prokaryotic or eukaryotic sequences.

Typically, 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. Such amino acid residues can be bioactive domains or assembly domains, such as the V domain or a foldon sequence such as

GYIPEAPRDGQAYVRKDGEWVLLSTFL (Du et al. (2008) Applied Microbiology and

Biotechnology 79(2) , 195-202.)

"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, 30°C, 31 °C, 32°C, 33°C, 34°C, or 35°C to about 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, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C or 36°C; that self-assembles into a triple helix in acidic, neutral, and/or basic solutions; that has thermal stability that is about the same or better than collagen; that is more collagenase-resistant than collagen; and/or that is modified to increase availability of lysines, and/or tyrosines and/or tryptophans and/or phenylalanines and/or histidines and/or glutamines for crosslinking and cysteines for conjugation to other polymers and peptides. Generally such collagen-like proteins have at least 70% sequence identity with one or more of the sequences shown in Figures 1 , 2A, or 2B.

Polypeptides, Nucleic Acids, Cells, Compositions, Products

As described above, provided herein are modified B subdomains of collagen-like proteins. Naturally occurring collagen-like proteins are derived from bacteria and do not have attributes that make them useful as a collagen replacement. However, as shown in the examples below, 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.

In the modified collagen-like proteins or subdomains thereof, it will be understood that the absolute number or position of the modified amino acids (e.g., lysines, tyrosines tryptophans, phenylalanines, histidines and/or glutamines) 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.

Thus, provided herein in is a 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: MNHKVHMHHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLL T YLQEREQAENSWRKRLLKGIQDHALDGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGE QGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGKDGERGPVGPAGPKGEQGPQGL PGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGK DGERGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGD RGETGPKGPKGERG E AG PAG KD G E RG P VG PA (SEQ ID NO: 1) or a fragment thereof. In certain aspects, the polypeptide is identical to SEQ ID NO: 1 or a fragment thereof. In aspects, the fragment described herein represents a single triple-helix motif of the polypeptide, which may be then repeated 1 or more times as described herein.

In aspects, the sequence

MNHKVHMHHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMT KLLT 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.

The desired biological functionality of the polypeptides described herein may be facilitated by the addition of one or more moieties. Examples of such moieties include, but are not limited to peptides, carbohydrates, small molecules, drugs, antibodies, PEG-based compounds, toxins, dyes, imaging agents or binding sequences.

For example, the 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 α2β1 integrin or an α3β1. 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.

In specific aspects, 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. Typically, the bioactive cell binding domain is linked to the C-terminal end of the polypeptide with an optional linker sequence. In typical aspects, the linker sequence comprises either a short sequence such as (GSTSGSGT)n, wherein n is an integer or a long sequence such as

(GSTSGSGKPGSGEGSTKGT)n (Whitlow et al. (1993), Protein Eng., 6, 8, 989-995).

In aspects, 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, and/or the elastomeric domain comprises at least one of VPGVG, (AG) ₃ EG, GYSGGRPGGQDLG, GGFGGMGGGS, PGQGQQ, and GYYPTSPQ. Further, 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.

In aspects, the fragment of the polypeptide described herein may comprise the sequence

GPXGEQGPQGLPGKDGEAGAQGPAGPMGPAGEXGEKGEPGTXGAKGDRGETGPXGPX 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.

In other aspects, the 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.

In other aspects, the 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.

These sequences may be repeated multiple times, such that there are 1 , 2, 3, 4, 5, 6,

7, 8, 9, 10 or more adjacent copies of the sequence. Typically, there are at least three copies of the sequence.

In aspects, 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.

As noted above, the 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.

The 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. There are 18,874 collagen-like proteins annotated in bacteria according to a Uniprot search conducted by Lukomski et al. ((2017) Molecular Biology 103(6), 919-930). Any of these may be modified and used as described herein.

For example, 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.,

Mycobacterium gilvum, Mycobacterium tuberculosis, Mycobacterium vanbaalenii,

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,

Dechloromonas aromatica, Polaromonas naphthalenivorans, Ralstonia eutropha, Ralstonia metallidurans, Ralstonia pickettii, and Rhodoferax ferrireducens), Cyanobacteria (e.g., Cyanothece species, Synechocystis species, Trichodesmium erythraeum), Deinococcus (e.g., Deinococcus radiodurans), Deltaproteobacteria (e.g., Anaeromyxobacter

dehalogenans), 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, Pseudomonas aeruginosa, Pseudomonas entomophila, Pseudomonas putida, Psychrobacter

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).

In typical aspects, 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. (2009) Research in Microbiology 160(10) 792-799); or sub-sequences from Methylobacterium sp 4-46, Rhodopseudomonas palustris, Solibacter usitatus, or Clostridium perfringens (Xu et al. (2010) Biomacromolecules 11 (2), 348).

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.

The polypeptides described herein 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. For example, unlike collagen, high concentration collagen-like polypeptide solutions can be crosslinked at acidic and neutral pH to fabricate thermally stable, transparent hydrogels. The presence of key functional groups throughout the collagen-liked polypeptide sequence provides the user with targets (e.g., primary amines, aromatics, and/or glutamine) to facilitate inter- and intra-molecular crosslinking by a variety of methods. For example, by varying the molar equivalent ratio of a chemical crosslinker such as EDC or DMTMM to the lysine groups the user can tune the physical properties (e.g. tensile strength, modulus) of the crosslinked hydrogel. 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.

Alternatively, 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. For example, it has been previously demonstrated that in the presence of 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). Alternatively, 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. As 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. August 6; 24(29): 4615- 4624). It is also possible to vary the lysine to glutamine ratio to crosslink the material to varying degrees in the presence of calcium cations and the appropriate enzyme. Similarly, through experimentation the ratio of crosslinking reagents to functional groups can be optimized to tune cross-linked material properties.

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.

Experimental Examples

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

The following examples do not include detailed descriptions of conventional methods, such as those employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids, or the introduction of plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications including Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, which is incorporated by reference herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the typical aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Materials

1 -Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), 4,6-dimethoxy-1 ,3,5-triazin- 2-yl)-4-methylmorpholinium chloride (DMTMM), sodium bicarbonate (NaHC0 ₃ ), 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 (PBS) 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

(Manassas, VA, USA). 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/DEAD™

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 Vector construction

Recombinant bacterial collagen-like sequence (HyColl) was composed of a modified, triple-repeated sub-domain sequence from bacteria collagen CL domain with the known N- terminal V domain. The bacteria codon-optimized HyColl sequence was cloned into pCold III (Takara Bio Inc.) using Nde I and BamH I (New England Biolabs).

Recombinant Protein Expression in E. coli

The 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 μg/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. Large scale expression (5 L) was performed at 37°C and with 35% of oxygen saturation using a BioFlo 1 10 Modular Benchtop Fermentor (New Brunswick Scientific Co. Inc., Enfield, USA). The media for 5 L protein expression consisted of the following: 50 g glucose, 100 g yeast extract, 10 g (NH^HPC , 33.75 g KH ₂ P0 ₄ , 4.25 g citric acid, 3.5 g MgS0 ₄ - 7H ₂ 0, 25 ml trace metals, 500 mg Ampicillin (100 mg/ml), and 0.5 ml antifoam. 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.

Recombinant Protein Purification

After being harvested by centrifugation, the pellet was re-suspended in His-tag purification column binding buffer (20 mM sodium phosphate buffer pH=7.4, 500 mM NaCI, 10 mM imidazole) containing 0.25 mg/ml lysozyme (Thermo Fisher Scientific, Inc.) and 1 mM (phenylmethylsulfonyl fluoride) PMSF (Sigma). Cells were then subjected to three freeze- thaw cycles and sonicated for 2 min. The suspension was incubated by stirring at room temperature for 30 min after DNasel were added with final concentration of 20 μg/ml. 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 μηι filter membrane before purifying with columns packed with IMAC Sepharose™ 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. Pure proteins were then loaded into Spectra/Pro dialysis tubing (12-14 K MWCO) (Spectrum Laboratories, Inc.), dialyzed again MiliQ water (above 15 mQ) for 72 h, and then lyophilized and stored at 4°C until use.

Preparation of EDC and DMTMM Crosslinked HyColl and HyColl-RGD Hydrogels

Pre-determined aliquots of HyColl, HyColl-RGD or porcine collagen solution (used as a comparator) 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.

Preparation of Photo-Crosslinked HyColl-Tyrosine-RGDS Hydrogels

Pre-determined amounts of lyophilized HyColl-Tyrosine-RGDS or HyColl (used as a comparator) 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.

For a 10% (w/v) protein solution, 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.

*40mM SPS is used when SPS is used in the absence of Ru(bpy) ₃ CI ₂

**20mM SPS is used when SPS is used with 2mM Ru(bpy) ₃ C

Preparation of Enzyme Crosslinked HyColl-Tyrosine-RGDS Hydrogels

Pre-determined amounts of lyophilized HyColl-Tyrosine-RGDS or HyColl (used as a comparator) 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%) were added to their respective protein solutions at room temperature and thoroughly mixed. For a 10% (w/v) protein solution sample, volumes are provided as in Table 2. 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 2. Sample volumes for HRP Crosslinked HyColl-Tyrosine-RGDS solution.

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 μηι I.D. χ 100 mm fused silica analytical column packed in-house with 3 μηι ReproSil-Pur C18 beads (100 A; Dr. Maisch GmbH, Ammerbuch, Germany) at a flow rate of 1 .5 μΙ_/ηιίη for 15min. Then the flow of 20 μΙ_/ηιίη from HPLC was split into 200 nL/min to perform the peptide separation. Gradient elution was set as 5-50% buffer B in 40 min, followed by 2 min 100% buffer B and 10 min 2% buffer B to re-equilibrate for the next run.

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. The full mass was scanned in Orbitrap analyzer with R = 60,000 (defined at m/z 400). To improve the mass accuracy, all the measurements in the orbitrap mass analyzer were performed with a real time internal calibration by the lock mass of background ion 445.120025.

Molecular weight was calculated using adjacent charge states as follows:

X= (MW + z)/z

Y = (MW + Z + 1)/(z + 1)

Zx = (Y-1)/(X-Y)

MW = (X * Zx) - Zx = (Y * Zy) - z _y where MW is the molecular weight of protein and z is the charge state.

Circular Dichroism (CD)

CD spectra were measured for 10 μΜ 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. 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. Thermally induced denaturation of the HyColl, HyColl-RGD and HyColl-Tyrosine-RGDS solution was 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.

Data were converted into mean residue molar ellipticity [Θ] defined by the equation: where 0 _Ob s(A)∞ (A _L (A) - A _R (A)) is the observed ellipticity in mdeg, c is the protein

concentration in M, / is the path length in cm, and n is the number of amino acid residues in a protein.

Fourier Transform Infrared Spectroscopy (FTIR)

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).

Fluorescence Spectrometry

Fluorescence studies were conducted using a Cytation 3 fluorescence

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.

Thermal Properties: Differential Scanning Calorimetry (DSC)

DSC studies were carried out on 100 μΜ HyColl, HyColl-RGD and HyColl-Tyrosine-

RGDS solutions at pH 4, pH 7 and pH 10 and 10 μΜ 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.

Mechanical Property Measurements

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 ^χ 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. First, strain amplitude sweeps were conducted at a fixed frequency (ω = 1 s ^-1 ) to map out the linear response regime. Next, frequency sweeps (over a range of 0.01 < ω < 10 s ^-1 ) at low stain amplitude (γο=10 ^-3 ) were conducted before and after large shear amplitude deformation (γο=1) in order to characterize the linear storage and loss moduli and the recover from non-linear deformation.

In vitro Degradation

Briefly, hydrated EDC/NHS cross-linked hydrogels (approximately 50 mg) were placed in vials containing 5 mL of a 5 U/mL collagenase in a PBS solution (Type I

Collagenase from Clostridium histolyticum, 318 U/mg solid, Sigma-Aldrich, Oakville, Ontario), refreshed every 8 hours. The vials were incubated in an oven at 37°C. The gels were weighed at different time intervals after removal of surface water through blotting. The residual mass of the hydrogels was tracked as a function of time, relative to their initial hydrated weight.

Scanning Electron Microscopy (Cryo-SEM)

Low temperature scanning electron microscopy (Cryo-SEM) measurements of EDC/NHS crosslinked HyColl hydrogels were carried out in a Tescan (Vega II - XMU) with a cold stage sample holder operating at -45°C. Backscattering detector (BSE) and secondary electron detector (SED) were used for collecting sample signals. A 2x2 mm material piece was cut and imaging was done from a cross section of the material from a section that was not sliced. Sample was gently blotted and temperature rapidly decreased under vacuum. In all cases fast speed scanning was used to minimize sample burning.

Cell Study

C2C12 mouse myoblast cells were cultured in a humidified incubator at 37 °C and

5% C0 ₂ in a DMEM medium containing 300 mg/L L-glutamine, 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. The experiments were performed with Serum-free medium with 100 U/ml penicillin and 100 mg/ml streptomycin to minimize competitive adsorption effects occurring for serum proteins. C2C12 Cells were cultured on polystyrene plates coated with 25 μΜ of HyColl and Hycoll-RGDS protein solution (Figure 46). The responses of C2C12s 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. 25uM protein solutions 10mM phosphate buffer at pH 7.5 were first filtered through 0.22 μηι syringe filters, and then triplicate wells were coated by passive adsorption overnight at 4 °C and rinsed excessively with PBS. Approximately 150 cells/mm ² cells in serum-free media were seeded into each well for 24 h, and phase contrast microscopy images were taken with an inverted microscope (Olympus, Japan).

The samples were also prepared using the same method for the WST-1 Cell

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 μΙ_ 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).

To evaluate the biofunctionality of both the photo- and the enzyme crosslinked HyColl-Tyrosine-RGDS hydrogels, 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. 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

Manufactured that Possesses High Solubility in Acidic, Physiologic and Basic pH.

Production in a shake flask gives yields of product < 1 gram per litre; however increased yields can be achieved by transferring the process to a stirred tank bioreactor. It is also possible that other vectors could give better commercial yields than observed with the pCold system (Yu, Z. et al. (2014) Journal of Structural Biology 186,451 -461). It has been previously reported that V-CL is soluble in 0.1 M acetic acid at 4°C. V-CL is only partially soluble (1 mg/ml) in PBS (pH 7.4) and becomes increasingly less soluble, forming a precipitate as the concentration increases to 2 mg/ml and the temperature increases to 24°C (Yoshizumi, A. et al. (2009) Protein Science 18, 1241 -1251). 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). In comparison 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),

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.

SDS-PAGE was used to confirm protein purity. As shown in Figure 4, 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.

Liquid Chromatography-Mass Spectrometry (LC-MS) was used to confirm the molecular weight of the HyColl. As shown in Figure 5, LC-MS gives a calculated molecular weight of 32875.64 Da comparable to the theoretical value of 32 kDa.

FTIR spectra as shown in Figure 6 displays the amide I band typically found in the range of 1600 and 1700 cnr ¹ which is the most intense absorption band in proteins and is characteristic of collagen and collagen-like material. It is primarily governed by the stretching vibrations of the C=0 and C-N groups. The exact band position is determined by the backbone conformation and the hydrogen bonding pattern. 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 μΜ 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 [Θ] of HyColl and HyColl-RGD respectively at 20°C as a function of wavelength λ 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. These figures confirm that HyColl and HyColl-RGD are stable in solution at about 5 to about 36°C at physiologic pH and retain a triple helix structure in acidic, physiologic and basic pH environments. The net observed spectra for the proteins in each buffer is a result of the opposing contributions from the a-helix structure of the V domain and the triple helix structure of the BBB domain of the protein i.e. the 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%) β-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.

Thermally-induced denaturation of the protein was examined by monitoring the mean residue molar ellipticity [Θ] as a function of increasing temperature at 220 nm. Plots for HyColl are provided in Figures 9 to 13). When 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 (ΔΤ < 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. Biochem 92, 563-567). 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.

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 (ΔΤ < 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.

Table 4: The thermal stability for HyColl-RGD at acidic, neutral and basic pH as determined

Example 5: Thermal Denaturation of HyColl is a pH Dependent, Reversible Process.

Refolding of the HyColl protein in different pH buffers was examined by monitoring the CD spectra at 220 nm after heating samples to 55°C followed by cooling to 15°C. The thermal unfolding of the protein in citric acid/Na ₂ HP0 ₄ buffer (pH 4 and pH 7) and

Na ₂ HP0 ₄ /NaH ₂ P0 ₄ (pH 7) is readily reversible. As shown in Figures 9 to 11 , a substantial amount of the original CD signal is regained during the cooling process, indicating the protein is capable of refolding. HyColl in MES buffer (pH 4.5) and NaHC0 ₃ /Na ₂ C0 ₃ buffer (pH 10) as shown in Figures 12 and 13 did not refold.

HyColl-RGD in citric acid/Na ₂ HP0 ₄ buffer (pH 4 and pH 7) and protein in

Na ₂ HP0 ₄ /NaH ₂ P0 ₄ (pH 7) and PBS (pH 7.4) refolded as shown in Figures 14 to 17 while HyColl-RGD in NaHC0 ₃ /Na ₂ C0 ₃ buffer (pH 10) as shown in Figure 18 did not refold. It has been previously shown that the V domain is important for refolding isolated collagen domains (4).

In order to further study the thermal behaviour of the protein, refolding was examined for the protein in citric acid and Na ₂ HP0 buffer (pH 7) by monitoring the CD spectra from 190 to 250 nm while increasing the temperature from 20°C to 50°C, and then cooling to 5°C as shown in Figure 19. The signal at 220 nm decreases as the temperature increases. At 40°C the spectra no longer show the characteristic features of collagen triple helix spectra (e.g. 220 nm maximum), but rather a random coil structure suggesting that the triple helix has unfolded. After cooling for 24 hours at 5°C, 99% of the original CD signal is regained demonstrating that the protein has refolded. It has been previously shown that isolated V domain trimerizes and is able to refold very quickly within the time that the sample is cooled (2).

Example 6: Similar Thermal Transitions are Seen by Micro-Differential Scanning Calorimetry (Micro-DSC).

Micro-DSC studies were carried out for the HyColl and HyColl-RGD in acidic, neutral and basic pH buffers. The scans obtained for 100 μΜ HyColl and 10 μΜ HyColl are shown in Figures 20 to 24 and Figures 25 to 29 respectively in different pH buffers with the heat flow rate as a function of the temperature. The scans obtained for 100 μΜ HyColl-RGD and 10 μΜ HyColl-RGD are shown in Figures 30 to 34 and Figures 35 to 39, respectively. The thermal stability data by micro-DSC for both HyColl and HyColl-RGD is summarized in Tables 5 and 6.

Denaturation temperatures (Tm) in the range of 35.2°C to 37.4°C for 100 μΜ 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 μΜ of HyColl shows the similar thermal scans with 100 μΜ 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.

Table 5: The thermal stability of HyColl solutions in acidic, neutral and basic pH buffers

Table 6: The thermal stability of HyColl-RGD solutions in acidic, neutral and basic pH

As previously shown by CD, 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). At 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.

Initial crosslinking experiments were done to demonstrate that covalently crosslinked hydrogels that are thermally stable at physiologic temperature (37°C) can be fabricated. Hydrogels were fabricated as shown in Figure 40 using N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC), a zero-length crosslinker used to crosslink collagen in the presence of N-hydroxysuccinimide (NHS) in MES (pH 4.5) buffer. However, efficient EDC activation of carboxylic acids occurs in a narrow acidic pH range (i.e. pH 4.5 - 5.5). Given that the material demonstrated good solubility and thermal stability at both physiologic and basic pH we have also fabricated hydrogels using the non-pH dependent crosslinker, 4- (4,6-dimethoxy-1 ,3,5-triazin-yl)-4-methylmorpholinium (DMTMM) to fabricate HyColl hydrogels.

DSC was used to determine the temperature at which the 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. It is interesting to note that the thermal stabilities of 10% (w/w) solutions of HyColl and HyColl-RGD (37.58°C and 39.39°C respectively) in PBS are superior to that of an equivalent porcine derived type I atello- collagen solution which was determined to be 35.51 °C using the same instrumentation.

Example 8: Mechanical Properties Equivalent to Collagen Hydrogels Can Be Achieved.

The tensile strength, moduli and elongation at break of HyColl hydrogels prepared from 10% (w/w) solutions and crosslinked with EDC/NHS in MES (pH 4.5) buffer are shown in 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. In this case, the collagen solution pH was approximately 2 and required pH adjustment using sodium hydroxide to enable EDC/NHS crosslinking. For the HyColl, no pH adjustment was required.

In addition, dynamic shear moduli (storage and loss) in response to applied oscillatory strain were measured for HyColl hydrogels prepared from 5 and 10% (w/w) solutions crosslinked with EDC/NHS in MES (pH 4.5) buffer with a crosslinking ratio of 0.5. Figure 41 shows a strain sweep measurement of the storage (G ^' ) and loss (G ^" ) moduli as a function of strain amplitude γ ₀ at a fixed frequency of ω = 1 s ¹ . The data indicate linear response well beyond 10% strain amplitude and a strain softening transition occurring near Yo=25%. Figure 42 shows a frequency sweep measurement at low stain amplitude (γο=0.1%) over a range of frequencies, 0.01 < ω < 10 s . The solid and open squares show data measured on previously unperturbed sample, while the solid and open triangles show data obtained after the application of a large shear amplitude deformation (γ ₀ = 100%). In all cases, 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.

In vitro HyColl hydrogel degradation by collagenase was evaluated by monitoring the residual mass percentage of the hydrogel as a function of time. For degradation property comparison, a porcine derived type I atello-collagen hydrogel was prepared using the equivalent concentrations and EDC crosslinking ratio. As shown in Figure 43, after 20 hours the porcine type I hydrogel had lost over 80% of its original mass while the HyColl hydrogel had maintained 97% of its original mass. After 48 hours, 6% of the porcine type I hydrogel mass remained compared to the HyColl hydrogel, which had retained 33% of its original mass, demonstrating that HyColl hydrogels are more resistant to collagenase compared to a porcine collagen equivalent. Example 10: Cryo-SEM Images of Crosslinked HyColl Hydroqels Show a Honeycomb Structure.

Low temperature scanning electron microscopy (Cryo-SEM) images of a section of an EDC/NHS crosslinked HyColl hydrogel are shown in Figure 44. The image shows a typical honeycomb like structure comprised of lamellae with fine interconnecting fibrils.

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.

Several variants were constructed and kept as glycerol stock including the variant HyColl-DGEA with spacer GSTSGSGT and cell binding domain DGEA (Figure 45A), variant HyColl-MLPHHGA with spacer GSTSGSGT and hydroxyapatite binding domain MLPHHGA (Figure 45B), variant HyColl-Tyrosine-RGDS (modified threonine with tyrosine) with spacer GSTSGSGT and cell binding domain RGDS (Figure 45C) and variant HyColl-Tyrosine- RGDS-Vcut (modified threonine with tyrosine) and inserted with cleavage tag LEVLFQGP between the V domain and B domain, with spacer GSTSGSGT and cell binding domain RGDS (Figure 45D). For the variant with the insertion of the cleavage tag, other 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 μΜ 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 [Θ] of HyColl -Tyrosine-RGDS at 20°C as a function of wavelength (λ) 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.

Thermally-induced denaturation of the protein was examined by monitoring the mean residue molar ellipticity [Θ] as a function of increasing temperature at 220 nm.

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. The thermal unfolding of the protein in citric acid/Na ₂ HP0 ₄ buffer (pH 4), PBS(pH7.4) and

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.

Micro-DSC studies were carried out for the HyColl-Tyrosine-RGDS in acidic, neutral and basic pH buffers (Figure 52-55). The similar results were observed for HyColl-Tyrosine- RGDS with the denaturation temperatures (Tm) in the range of 34.9°C to 36.2°C (Table 10). The smaller secondary peaks may be due to soluble aggregates that are reversible with time.

Table 10: The thermal stability of HyColl-Tyrosine solution in acidic, neutral and basic pH

Example 12: Adhesion and Viability of Cells on Protein-Coated Surfaces

Initial in vitro cell studies were performed to look at the response of adhesion- dependent C2C12 cells on surfaces coated with either HyColl or HyColl-RGD. Good cell morphology and spread was observed on the surfaces coated with HyColl-RGD, while the cells maintained a round morphology and did not attach as expected to the surfaces coated with HyColl (Figure 56). The effect of RGD (RGDS) ligand density on cell adhesion and spreading was further evaluated. Figure 57 shows representative images of cell adhesion and morphology on the surfaces coated with increasing ratios of Hycoll-Tyrosine-

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. The metabolic activity of 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.

Example 13: HyColl-Tyrosine-RGDS Can Be Photo-Crosslinked to Produce Thermally Stable Transparent Hydrogels

Initial photo-crosslinking experiments were done to demonstrate that photo- crosslinked hydrogels that are thermally stable at physiologic temperature (37°C) can be fabricated using visible light. Hydrogels were fabricated using the well-known metal-ligand catalyst, Ru(bpy) ₃ CI ₂ in the presence of SPS. Solutions were exposed for 30 seconds to a 455 nm light source providing 28 mW/cm ² to the sample. Of particular note, HyColl-Tyrosine- RGDS can be photo-crosslinked in the absence of the catalyst. For example, hydrogels were fabricated using SPS only and APS only respectively. In both cases, solutions were exposed for 30 seconds to a 455 nm LED light source providing 28 mW/cm ² to the sample. An SPS only photo-crosslinked hydrogel is shown in Figure 59. The emission spectrum with an excitation wavelength of 315 nm for this hydrogel showing the emission maximum at 410 nm characteristic of the di-tyrosine bond is shown in Figure 60. 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, used as the comparator, remained as a viscous solution following light exposure.

DSC 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. The thermal stability increased from 36.64°C to 57.09°C upon photo-crosslinking in the presence of SPS alone exposed to blue light,to 49.95 °C upon crosslinking in the presence of APS alone exposed to blue light and to 44.10 °C in the presence of SPS alone exposed to white light demonstrating that HyColl-Tyrosine-RGDS can be photo-crosslinked to produce thermally stable transparent hydrogels. It is interesting to note that the thermal stability of a 10% (w/w) solutions of HyColl-Tyrosine-RGDS in PBS are superior to that of an equivalent porcine derived type I atello-collagen solution which was determined to be 35.51 °C using the same instrumentation.

Table 1 1. Thermal Stabilit of Photo-crosslinked H Coll-T rosine-RGDS H dro els

* 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

Initial crosslinking experiments were done to demonstrate that enzyme crosslinked hydrogels that are thermally stable at physiologic temperature (37°C) can be fabricated using HyColl-Tyrosine-RGDS. Hydrogels crosslinked using the well-known enzyme horse radish peroxidase in the presence of hydrogen peroxide are shown in Figure 62. HRP crosslinked gels did not have the mechanical strength as was achieved using the photo- crosslinking approach and therefore photos were taken of the gel in the dish. Increasing the ratio of units of HRP:tyrosine is expected to improve the mechanical properties of the fabricated hydrogel.

DSC was used to determine the temperature at which the enzyme crosslinked

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.

The bio-functionality of both the photo- and enzyme crosslinked HyColl-Tyrosine- RGDS hydrogels were evaluated with C2C12 cells. C2C12 cells remained viable and showed good spreading after 24 hours incubation on the photo crosslinked hydrogels (Figure 64) as well as the enzyme crosslinked hydrogels (Figure 65). The results

demonstrate that the 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.

Modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art and such modifications are within the scope of the present invention. Furthermore, all references cited herein are incorporated by reference in their entirety.