Decorating a Blank Slate Protein Hydrogel: A General and Robust

Sep 27, 2017 - Xiaotian LiuXin YangZhongguang YangJiren LuoXiaozhen TianKai LiuSongzi KouFei Sun. ACS Applied Nano Materials 2018 1 (4), 1579- ...
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Decorating A Blank Slate Protein Hydrogel: A General and Robust Approach for Functionalizing Protein Hydrogels Xiaoye Gao, Shanshan Lyu, and Hongbin Li Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01369 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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Decorating A Blank Slate Protein Hydrogel: A General and Robust Approach for Functionalizing Protein Hydrogels Xiaoye Gao,1# Shanshan Lyu1,2# and Hongbin Li1* 1

Department of Chemistry

University of British Columbia Vancouver, BC V6T 1Z1 Canada 2

State Key Laboratory of Organic-Inorganic Composite Materials Beijing University of Chemical Technology, Beijing, 100029 P. R. China

#

These authors contribute equally to this work.

*To Whom Correspondence Should Be Addressed ([email protected]).



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Abstract Protein hydrogels constructed from recombinant proteins have attracted increasing interests for fundamental biological studies as well as applications in biomedical engineering field. In such protein hydrogels, biochemical and physical properties of protein hydrogels are often coupled with each other, making it challenging to investigate the individual effect of chemical and physical cues on cells. Moreover, laborious engineering is often required to incorporate different protein ligands into such hydrogels. To address these challenges, functionalizing a blank slate protein hydrogel is an attractive approach. However, conjugating ligands to such a blank slate protein hydrogel is challenging, as existing bioconjugation methods developed in synthetic polymer hydrogels cannot be readily adapted for protein hydrogels, significantly impeding the use of this approach in the field. Here we report a facile, general and robust method, which is based on the SpyCatcher-SpyTag chemistry, to covalently functionalize the “blank slate” of protein hydrogels using genetically encoded interacting partners. We demonstrate that this novel method enables covalent conjugation of a wide variety of ligands, including full length intact folded proteins, to a blank slate protein hydrogel, and allows for the decoupling of biochemical and physical properties of hydrogels from each other and investigating the individual effect of biochemical and mechanical cues on cell behaviors. To our best knowledge, this is the first general approach enabling functionalization of protein hydrogels, and we anticipate that this novel approach will find a broad range of uses in protein-based biomaterials for applications in biomedical engineering.



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Introduction Hydrogels are polymer networks that swell in water. They have tissue-like water content, tunable chemical, physical and mechanical properties, making them ideal materials for a myriad of applications in the biomedical field, ranging from contact lenses, tissue engineering scaffold, target drug delivery vehicle and cell culture media.1-3 In biological studies, polymer hydrogels are often used as biomimetic extracellular matrices to provide biochemical and physical cues that are found in the natural extracellular matrices. To fulfill this purpose, a hydrogel “blank slate” is often constructed from bio-inert polymers, such as polyethylene glycol and alginate, followed by conjugation of biochemical ligands to functionalize the “blank slate”.4-6 Many successful conjugation methods have been developed to greatly facilitate the engineering of such functionalized polymer hydrogels, such as click chemistry.7-14 However, biochemical ligands conjugated to the hydrogel blank slate using such methods are typically limited to relatively small ones, such as the RGD sequence. Conjugating functional, intact protein domains into such hydrogels remains challenging.15 Compared with synthetic polymer-based hydrogels, hydrogels constructed from engineered recombinant proteins have attracted increasing interests over the last two decades,16-18 owing to the ability to precisely control protein sequence, molecular weight and folded structure (both secondary and tertiary), as well as their high potential for biomedical applications. It also becomes possible to incorporate intact functional protein domains into hydrogels to evaluate the influence of such intact biochemical ligands on cells in biological studies.17, 19-21 However, the incorporation of biochemical ligands into protein polymers often requires laborious engineering of new protein building blocks, and such new protein polymers often lead to the change of the physical properties of the resultant protein hydrogels. In addition, because of the coupling of functional protein ligands with the physical properties of protein hydrogels, it is challenging to investigate the individual effect of chemical and physical cues on cells.22, 23 A potential approach to address this challenge is to employ the blank slate approach used in polymer hydrogels. However, there is no general approach for approach for functionalizing blank protein hydrogel slate, and conjugation methods developed for synthetic polymer hydrogels are often challenging to implement in protein hydrogels.15 Based on the well-developed SpyCatcher-SpyTag chemistry,24, 25 here we report a facile,



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general and robust method to functionalize the “blank slate” of protein hydrogels using genetically encoded interacting partners. This new approach enables the decoupling of biochemical ligands from physical properties of hydrogels and thus allows for investigating the effect of biochemical and mechanical cues on cell behaviors. Materials and Methods Protein engineering The genes encoding GRG5RG4R, G-Sc and G-St, which were constructed in the expression vector pQE80L as described previously,26, 27 contain a 5’ BamHI, 3’ BglII and KpnI site. G-Sc, which was excised from pQE80L-G-Sc by restriction digestion using BamHI and KpnI, was subcloned into pQE80L-GRG5RG4R that was linearized by digestion with BglII and KpnI restriction enzymes to obtain pQE80L- GRG5RG4RGSc. ECFP-G-St, CYFPG-St and FnGSt were constructed in a similar fashion. Overexpression of the proteins was carried out in the Escherichia coli (E. coli) strain DH5α. The bacteria culture was grown at 37 °C in a 2.5% lysogeny broth (LB) containing 100 mg/L ampicillin. Protein over expression was induced with 1 mM isopropyl-1-β-D-thiogalactoside (IPTG) when the OD600 reached ~0.8. Protein expression continued for 4 h. The bacterial cells were harvested by centrifugation at 4000 rpm for 10 min, and lysed using a 2.5 mg mL-1 lysozyme solution. Proteins were harvested from the soluble fraction via Co2+-affinity chromatography; and the protein solutions were dialyzed against deionized water (changed every 7 h) for 72 h to remove salts. After dialysis, proteins were lyophilized. Molecular weights of proteins were determined using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Hydrogel construction and Coupling of St-containing proteins to hydrogels Lyophilized proteins GRG5RG4RGSc, ECFP-G-St, EYFP-G-St and FnGSt were weighted and dissolved in PBS to obtain protein solutions at desired concentrations. To prepare GRG5RG4RGSc-based hydrogels, lyophilized protein GRG5RG4RGSc was weighted and dissolved in PBS to obtain protein solutions at desired concentrations. Ammonium persulfate (APS) and [Ru(bpy)3]2+ were then added to the protein solution to final concentrations of 260 µM for [Ru(bpy)3]2+ and 50 mM for APS. The protein solution was cast



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into a custom-made Plexiglas mold (with an inner diameter of 8 mm, an outer diameter of 10 mm and a height of 3 mm), and irradiated for 10 min using a 150W fiber optical white light source placed 10 cm away from the mold. The resultant hydrogel was removed from the mold and stored in PBS buffer with 0.05% (w/v) sodium azide at 4 °C. For pre-hydrogelation coupling, St-containing protein was added to the GRG5RG4RGSc solution at a Sc-St molar ratio of 1:1 and allowed to react with GRG5RG4RGSc for 1 hr prior to the photochemical crosslinking reaction. For post-hydrogelation functionalization, the photochemically crosslinked GRG5RG4RGSc hydrogel was soaked in 3 mg/ml St-containing protein solution for 2 hrs to allow the Sc-St chemistry to occur. Then the hydrogel was rinsed in PBS buffer to remove unbound St-containing proteins. Fluorescence Microscopy Fluorescence imaging of hydrogels was carried out using a Nikon, Eclipse Ti inverted microscope equipped with an Andor EM-CCD camera. Two filter sets, one with a 436/20 nm excitation passband and 480/40 nm emission passband, and the other one with a 500/20 nm excitation passband and 535/30 nm emission passband, were used for observing ECFP and EYFP, respectively. Cell Culture and Cell Spreading Assay Human lung fibroblasts (HFL1) were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and cultured according to the recommended ATCC protocol. HFL1 were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Hyclone) and penicillin. Cells were maintained at 37 °C with 5% CO2, fed every 3 days and passaged according to protocol. Cell spreading assay were carried out in triplicate. FnGSt-functionalized GRG5RG4RGSc hydrogels were washed three times with fresh DMEM medium to remove any unconsumed reagents from the photochemical crosslinking reaction as well as unreacted FnGSt protein. 3000 HFL1 cells were seeded on top of each hydrogel. After 24 hrs of culturing, hydrogels were washed twice with pre-warmed, sterilized PBS (pH 7.2) to remove non-adherent cells. Actin filaments of adherent cells on all surfaces were stained with Alexa Fluor 488 phalloidin



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(Invitrogen) following the manufacturer’s protocol. After staining, fluorescence imaging was carried out using a Nikon, Eclipse Ti inverted microscope equipped with an Andor EM-CCD camera. Cell length was analyzed using ImageJ for comparison of cell behavior on different surfaces. Staining actin filaments alone allowed us to measure the cell areas without any difficulty. Although labeling the cell nuclei at the same time will help cell imaging experiments, it was not essential for our measurements. Rheology Measurements Viscoelastic properties of the hydrogels were characterized using a TA Instruments Discovery Hybrid Rheometer equipped with an 8 mm flat plate and a fiber optical illumination system. Frequency-dependent viscoelastic moduli (storage modulus G’ and loss modulus G”) were measured using a TA Instruments Discovery Hybrid Rheometer equipped with an 8 mm flat plate. The mechanical properties of GRG5RG4RGSc-based hydrogels were measured in frequency modes at room temperature at a 200 um gap with a strain of 2% by a frequencydependent sweep from 0.1 to 100 rad s-1.

Results and Discussion SpyCatcher-SpyTag chemistry provides a robust and facile conjugation method Our new approach is based upon the well-developed and efficient SpyCatcherSpyTag chemistry. The SpyCatcher-SpyTag chemistry was developed by Howarth and coworkers based on the discovery of a naturally occurring isopeptide bond in the second immunoglobulin-like collagen adhesin domain (CnaB2) of the fibronectin-binding protein (FbaB) of Streptococcus pyogenes.24, 25 SpyCatcher (138 aa) and SpyTag (13 aa) are two reactive protein partners engineered based on the sequences split from CnaB2. SpyCatcher and SpyTag can spontaneously react to reconstitute the intact folded CnaB2 domain and form a covalent isopeptide bond,24, 28 and the SpyCatcher-SpyTag chemistry has been used not only to develop protein tags, but also serves as driving forces to engineer protein-based hydrogels, protein polymers with defined topologies.20, 25, 26, 29 Previous studies from various research groups have established that SpyCacher and SpyTag can react with each other efficiently at ambient conditions and robustly regardless of their location ((N-, C-terminus or the middle of



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the sequence) or their fusion neighbors (folded globular domains or random coil like sequences) in a fusion protein.20, 25, 26, 29 Here, we employ SpyCatcher-SpyTag chemistry to enable efficient and robust protein hydrogels functionalization. Due to its small size,24 SpyTag can be easily incorporated into protein ligands that are of interest. Although most SpyCatcher-SpyTag reactions were carried out in solution, we speculate that SpyCatcher-SpyTag chemistry will also work inside the hydrogel as well as on the surface of the hydrogel. Thus, it should be feasible to use SpyCatcher-SpyTag chemistry to readily functionalize blank slate of protein hydrogels, if SpyCatcher is incorporated into the blank slate and SpyTag is incorporated into the protein ligands that are to be conjugated, or vice versa. Fig. 1 schematically shows the principle of this new design. To demonstrate the proof of principle of this novel approach, here we used GB1resilin-(GB1)5-resilin-(GB1)4-resilin (GRG5RG4R)-based protein hydrogel as a model system. GRG5RG4R is an engineered artificial elastomeric protein, which mimics the structure and

Figure 1. Schematics of functionalizing blank slate protein hydrogels via SpyCatcherSpyTag Chemistry. SpyCatcher-containing elastomeric proteins were photochemically crosslinked into protein hydrogels, which serve as the blank slate hydrogel. Then SpyTagcontaining functional ligands were conjugated to the protein hydrogel blank slate via the SpyCatcher-SpyTag chemistry.



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nanomechanical properties of the giant muscle protein titin, and has been used to construct protein hydrogels that mimic the passive elastic properties of muscle,27 where GB1 is the B1 IgG binding domain of protein G from Streptococcus,30 and resilin is a 15 aa consensus sequence found in the insect elastomeric protein resilin.31 The mechanical properties of GRG5RG4R-based hydrogels have been characterized in detail in our previous work.27 GRG5RG4R does not contain any cell binding sequence, and thus GRG5RG4R hydrogels do not support cell adhesion or cell spreading. Thus, GRG5RG4R-based hydrogels can serve as an ideal blank slate of protein hydrogel for functionalization. To demonstrate the principle of our approach, we introduced SpyCatcher domain to the C-terminus of GRG5RG4R as a conjugation site and obtained the protein GRG5RG4RGSc. Overexpression of GRG5RG4RGSc in E. Coli gave ~30 mg purified protein per liter of culture. Using well-developed Rutheniumcatalyzed photochemical crosslinking method (Fig. 2, top panel),31-33 we crosslinked the aqueous solution of GRG5RG4RGSc into transparent solid hydrogels (Fig. 2, bottom panel), which serves as our blank slate hydrogel. The blue fluorescence of the hydrogels under UV illumination originates from the dityrosine adducts, which are formed by the crosslinking of two tyrosine residues that are in close proximity and serve as the chemical crosslinking points of the hydrogel network.

Figure 2. GRG5RG4RGSc-based hydrogel blank slate. Top panel shows the schematics of the photochemical crosslinking chemistry used to engineer protein hydrogels. Bottom panel shows the photographs of GRG5RG4RGSc-based hydrogel under white light (left) and UV illumination (right). The blue fluorescence originates from dityrosine adducts, which serve as the chemical crosslinking points in the hydrogel.

To demonstrate the feasibility of functionalizing the blank slate hydrogel using the SpyCatcher-SpyTag chemistry, we first used fluorescent proteins as model systems (enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein(EYFP)). ECFP-G-St



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and EYFP-G-St were constructed to serve as protein ligands to be conjugated to the protein hydrogel blank slate.

Figure 3. Fluorescence microscopy images of GRG5RG4RGSc hydrogels functionalized by fluorescent proteins. To show the contrast, the edge of the hydrogels was imaged. Top panel corresponds to the images for ECFP-functionalized GRG5RG4RGSc hydrogels together with the control GRG5RG4RGSc with an excitation wavelength of 436 nm. Bottom panel corresponds to the images for EYFP-functionalized GRG5RG4RGSc hydrogels together with the control GRG5RG4RGSc with an excitation wavelength of 500 nm.

Two different methods were tested to functionalize the GRG5RG4RGSc-based hydrogels. One is to couple ECFP-G-St (or EYFP-G-St) to GRG5RG4RGSc in solution prior to the photochemical crosslinking reaction (pre-hydrogelation), and the other one is to couple ECFP-G-St to GRG5RG4RGSc-hydrogels (post-hydrogelation). Fig. 3 shows fluorescence photographs of the hydrogels. ECFP (or EYFP) functionalized GRG5RG4RGSc-hydrogels, either by pre- or post-hydrogelation, show strong cyan (or yellow) fluorescence (Fig. 3A-D). In contrast, the control GRG5RG4R hydrogel did not show any ECFP (or EYFP) fluorescence even after soaking in ECFP (or EYFP)-G-St. These results clearly demonstrate that the GRG5RG4RGSc-based hydrogels can be readily functionalized via SpyCatcher-SpyTag chemistry using SpyTag-fused full length protein ligands, and both methods (pre- or posthydrogelation) can lead to efficient labeling of the hydrogel, demonstrating the versatility of this novel conjugation method. It is of note that coupling ECFP-G-St to GRG5RG4RGSc



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hydrogels requires longer time to achieve similar fluorescence intensity than the prehydrogelation method, possibly due to the slow diffusion of ECFP-G-St into GRG5RG4RGSchydrogels. Since SpyTag is genetically encoded and can be easily fused with any protein (or peptide)-based ligand, the GRG5RG4RGSc-based hydrogels can, in principle, be functionalized by any protein ligand of interest to entail the hydrogel blank slate with any desired biological functionality. As a proof-of-principle experiment, we functionalized GRG5RG4RGSc-based hydrogels using SpyCatcher-SpyTag chemistry to make the hydrogel cell adhesive. The cell adhesive ligand we chose is the third fibronectin type III domain from the human extracellular matrix protein tenascin-C, which binds integrin and supports cell adhesion.21, 34 For this purpose, we engineered TNfn3- GB1-SpyTag protein (FnGSt), which serves as the protein ligand for conjugation.

Figure 4. FnIII domain-functionalized GRG5RG4RGSc-based hydrogels support the adhesion and spreading of fibroblast. A) GRG5RG4RGSc-based hydrogel blank slate does not support cell adhesion. B) Morphology of fibroblasts cultured on FnGSt-functionalized 15% GRG5RG4RGSc hydrogel. The cells are well-spread on the hydrogel surface.

GRG5RG4RGSc does not contain any cell adhesive ligand, and thus GRG5RG4RGSc-based hydrogels do not support cell adhesion or cell spreading. Indeed, after culturing human lung fibroblast on GRG5RG4RGSc hydrogels for 24 hrs, hardly any cell was found on the GRG5RG4RGSc hydrogel (Fig. 4A). To entail GRG5RG4RGSc hydrogel with



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cell adhesion properties, we reacted GRG5RG4RGSc with FnGSt at a Sc:St molar ratio of 1:1, and then carried out photo-crosslinking reaction to prepare a 15% GRG5RG4RGSc-based hydrogel. We then carry out cell spreading assay on these FnGSt-functionalized hydrogels using human lung fibroblast cells. After culturing for 24 hrs, fibroblast cells were fixed and permeabilized, and the cytoskeletal protein actin was fluorescently stained using Alexa fluor488-conjugated phalloidin. As shown in Fig. 4B, the fibroblast cells attached well to the FnGSt-functionalized hydrogel and displayed a spread morphology expected for adherent fibroblast cells,35, 36 demonstrating that Fn-functionalization successfully renders the GRG5RG4RGSc-based hydrogel cell adhesive. SpyCatcher-SpyTag chemistry-based conjugation method allows for independent tuning of the chemical and physical properties of the hydrogel Our results have clearly demonstrated that SpyCatcher-SpyTag chemistry offers a robust and efficient means to functionalize protein hydrogels using well-characterized protein hydrogels as a blank slate. Given the ease of fusing SpyTag to protein ligand, this novel approach has the potential to serve as a general protein hydrogel functionalization method. In this approach, the protein hydrogel blank slate is constructed using the photochemical crosslinking chemistry, and the SpyCatcher-SpyTag chemistry is used as a bioconjugation method to functionalize the protein hydrogel slate and is not involved in forming hydrogel network. This approach is conceptually different from those using SpyCatcher-SpyTag chemistry to engineer protein hydrogels,20, 26 where SpyCatcher-SpyTag chemistry is used to form crosslinked network and no protein hydrogel blank slate is involved. Beyond functionalization, this approach also offers the feasibility to tackle one of the challenges in protein hydrogels: how to decouple the effects of the biochemical and physical properties of hydrogels on cell behaviors. Taking collagen hydrogel22, 37 or our previously engineered FRF4RF4R-based hydrogel21 as an example, changing the concentration of collagen (or FRF4RF4R) will not only change the biochemical ligand density, but inevitably also change the physical properties of the hydrogel, such as the stiffness. Combining SpyCatcher-SpyTag chemistry with the hydrogel blank slate enables us to independently tune the biochemical and physical properties of hydrogels, thus helping decouple and elucidate the individual effect of biochemical and physical cues on cell behaviors. As a proof-of-principle,

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we carried out cell culture experiments to elucidate the effect of hydrogel stiffness on the spreading behaviors of fibroblast while keeping the density of biochemical ligand constant.

Figure 5. Viscoelastic properties of GRG5RG4RGSc-FnGSt-based hydrogels. A) Time evolution of the storage and loss modulus of GRG5RG4RGSc-FnGSt-based hydrogels during the photochemical crosslinking reaction. In these measurements, GRG5RG4RGSc concentration varied from 5% to 15% while the concentration of FnGSt remained constant. B) Storage and loss modulus of GRG5RG4RGSc-FnGSt-based hydrogels as a function of angular frequency (0.1 -100 Hz). The storage modulus of GRG5RG4RGSc-FnGSt-based hydrogels increased from ~9 kPa to ~30 kPa when the protein concentration increased from 5% to 15%.

We prepared GRG5RG4RGSc-FnGSt-based hydrogels at different GRG5RG4RGSc concentrations (5%, 10% and 15%) while keeping FnGSt concentration the same throughout. To do so, we first reacted GRG5RG4RGSc with FnGSt, and then carried out photocrosslinking reaction to prepare protein hydrogels. In all three hydrogels, the SpyTag concentration was kept the same and gave a Sc-St ratio of 1:1 in the 5% hydrogel (and thus 2:1 and 3:1 in 10% and 15% hydrogels, respectively). Since no hydrogel erosion was



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observed (data not shown), the actual FnGSt concentration in all three hydrogels should be very similar to each other and to the original value used preparing the hydrogels.

Figure 6. Morphology of fibroblast cultured on GRG5RG4RGSc-FnGSt hydrogels depends on the storage modulus of the hydrogel. A-C) Representative fluorescence microscopy images of fibroblast cells on FnGSt-functionalized GRG5RG4RGSc hydrogels (A: 5%, B: 10% and C: 15%). Actin filaments were stained with Alexa Fluor 488 Phalloidin. On 5% hydrogel, the number of adherent cells is low and a large number of fibroblasts remained round and unspread. On 15% hydrogel, the number of adherent fibroblast cells is the highest among the three hydrogels, and the cells were well-spread. D) The area of fibroblast cells adherent to the hydrogel surface. The average area of fibroblast cells is 19.6±15.3 µm2 (n=101), 21.8±16.5 µm2 (n=355), 29.7±20.5 µm2 (n=639) on 5%, 10% and 15% hydrogels, respectively.

Fig. 5 shows the rheology results for these three samples, where the storage modulus of the hydrogel increases from ~9 kPa to ~30 kPa when the GRG5RG4RGSc concentration increases from 5% to 15%, demonstrating the possibility of decoupling biochemical ligand density from the physical properties of the hydrogel. Since the biochemical ligand FnGSt was kept constant in all three hydrogels, the cell spreading behavior can thus be readily attributed to the change of stiffness of the hydrogels (Fig. 6). On the softest 5% hydrogel, the number of



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fibroblast cells is the lowest among the three samples, and many cells remained round and unspread. In contrast, on the hardest 15% hydrogel, the number of cells adherent to the hydrogel increased significantly, and most cells displayed an elongated and spread morphology (Fig. 6). And the cell area of fibroblast increased with the increasing of the hydrogel stiffness (Fig. 6D), suggesting that higher stiffness of the GRG5RG4RGSc-FnGSt hydrogel promoted cell adhesion and cell spreading of fibroblast. These results are consistent with previous findings that fibroblast cells tend to spread well on stiff gel surfaces,38-40 and corroborate the importance of mechanical cues on the behaviors of cells.

Conclusion By combining SpyCatcher-SpyTag chemistry with protein hydrogel blank slate, we have demonstrated a robust and efficient approach to functionalize protein hydrogels. Incorporating SpyCatcher domain into protein hydrogel blank slate and incorporating SpyTag into protein ligands, we demonstrated that the hydrogel blank slate can be readily functionalized with various protein ligands. The ligand that can be conjugated to protein hydrogels is not limited to small peptide, full length protein domains, for example green fluorescent protein and FnIII domain from tenascin, can be readily incorporated. Although only fluorescent proteins and FnIII domain were used to demonstrate the proof-of-principle, this approach enables one to conjugate essentially any protein ligand to protein hydrogels for a myriad of applications. For example, protein hydrogels can be easily decorated with protein growth factors to enable more complex studies than just FnIII domain-mediated cell spreading. This method also provides opportunities for incorporating non-peptide ligand to protein hydrogels. For example, DNA or RNA ligands can be conjugated to SpyTag via various chemistry, making it possible to readily label protein hydrogels with DNA/RNA ligands. Thus, this new method represents a general approach for functionalizing protein hydrogels. Furthermore, this method makes it possible to decouple biochemical ligand density from physical properties of hydrogels, and thus allows for detailed studies of the effect of biochemical ligand and physical cues on the behaviors of cells. We anticipate that this novel approach will find a broad range of applications in protein-based biomaterials.



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Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada. S. Lyu acknowledges the fellowship support from China Scholarship Council.



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