Engineering Protein Hydrogels Using SpyCatcher-SpyTag Chemistry

Subscriber access provided by Northern Illinois University

Article

Engineering Protein Hydrogels Using SpyCatcher-SpyTag Chemistry Xiaoye Gao, Jie Fang, Bin Xue, Linglan Fu, and Hongbin Li Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00566 • Publication Date (Web): 31 Jul 2016 Downloaded from http://pubs.acs.org on August 2, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Engineering Protein Hydrogels Using SpyCatcher-SpyTag Chemistry Xiaoye Gao1#, Jie Fang1#, Bin Xue2, Linglan Fu1 and Hongbin Li1* 1

Department of Chemistry

The University of British Columbia 2036 Main Mall Vancouver, BC, V6T 1Z1 Canada 2

Collaborative Innovation Center of Advanced Microstructures National Laboratory of Solid State Microstructure Department of Physics Nanjing University Nanjing 210093, P. R. China

*To whom correspondence should be addressed ([email protected]) #

These authors contribute equally.

1 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Constructing hydrogels from engineered proteins have attracted significant attention within the material sciences, owing to their myriad potential applications in biomedical engineering. Developing efficient methods to crosslink tailored protein building blocks into hydrogels with desirable mechanical, physical and functional properties is of paramount importance. By making use of the recently developed SpyCatcher-SpyTag chemistry, we successfully engineered protein hydrogels based on engineered tandem modular elastomeric proteins. Our resultant protein hydrogels are soft but stable, and show excellent biocompatibility. As the first step, we tested the use of these hydrogels as a drug carrier, as well as in encapsulating human lung fibroblast cells. Our results demonstrate the robustness of the SpyCatcher-SpyTag chemistry, even when the SpyTag (or SpyCatcher) is flanked by folded globular domains. These results demonstrate that SpyCatcher-SpyTag chemistry can be used to engineer protein hydrogels from tandem modular elastomeric proteins that can find applications in tissue engineering, fundamental mechano-biological studies, and as a controlled drug release vehicle.


Page 2 of 20

Page 3 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Introduction Owing to their biological friendliness, potential biomedical applications, and the ability to precisely control protein structure and function, engineered protein-based hydrogels have attracted considerable interest over the last two decades1-10. Many different protein interactions have been utilized to construct physically cross-linked protein hydrogels with varying physical and biological properties11-17, ranging from the most widely used coiled coil domain interactions11,13,15,18,19, to recently explored interactions that drive protein fragment reconstitution20. In parallel to such efforts, various chemical crosslinking strategies to construct chemically crosslinked protein hydrogels have been explored, offering more avenues towards tailoring the physical and functional properties of protein hydrogels21-28. Most protein hydrogels use non-globular proteins as their building blocks29. However, biological information is often encoded in full-length folded globular proteins. Constructing protein hydrogels that contain fulllength folded globular domains is imperative for achieving desired biological functions within protein hydrogels; despite this importance, efforts towards achieving this goal remain limited4,20,24,30-32. In our previous endeavors, we have started to explore the use of tandem modular proteins, which consists of individually folded globular domains arranged in tandem18,33, to construct functional bio-mimetic protein hydrogels4. Such proteins closely mimic naturally occurring tandem modular proteins that are widely distributed in the extracellular matrix as well as within cells. Additionally, highly efficient ruthenium-mediated photocrosslinking chemistry22,34,35 has enabled us to construct tandem modular protein-based hydrogels with tailored physical, mechanical and biological functions32,36. However, ammonium sulfate is essential in this photocrosslinking chemistry, and concerns remain over its use in biological applications owing to its potential cytotoxicity4,37. Further exploring and refining highly efficient and cyto-compatible crosslinking chemistry methods for constructing tandem modular proteinbased hydrogels is thus of great importance. Here we report the use of the recently developed highly efficient SpyCatcher-SpyTag chemistry to engineer tandem modular protein-based hydrogels. The SpyCatcher-SpyTag was first developed by Howarth and coworkers as a peptide tag38-40. They reported that the second immunoglobulin-like collagen adhesion domain (CnaB2) from the fibronectin binding protein (FbaB) of Streptococcus pyogenes contains a natural intra-molecular iso-peptide bond formed spontaneously between two adjacent residues Lys31 and Asp117 which are located in two


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

neighboring β-strands (Fig. S1)39. Howarth and coworkers were able to split CnaB2 into two reactive fragments: a 13-amino-acid peptide tag (named SpyTag), and a C-terminal fragment containing 138 residues (named SpyCatcher)38. By optimizing the sequences, SpyCatcher and SpyTag can spontaneously react to reconstitute the intact folded CnaB2 domain41. Under mild conditions, the SpyCatcher-SpyTag chemistry proceeds efficiently at temperatures ranging from 4 ºC to 37 ºC and does not require additional chemical reagents or catalysts38,42,43. This reaction has been used to engineer unusual nonlinear elastin-like protein polymers43. Recently, Tirrell and coworkers have demonstrated the utility of this reaction in constructing elastin-like protein-based hydrogels28. To fully explore the potential of SpyCatcherSpyTag chemistry in the construction of diverse protein hydrogels and develop this new chemistry into a robust chemical crosslinking methodology, here we explore the feasibility of using SpyCatcher-SpyTag chemistry to engineer tandem modular protein-based hydrogels. Instead of using non-structured elastin-like protein sequences, we used folded globular domains (GB1 and FnIII domain) as building blocks. We engineered tandem modular proteins that contain either multiple SpyCatcher or SpyTag sequences, and used them to construct protein hydrogels. We found that such SpyCatcher and SpyTag-containing tandem modular proteins can rapidly react with each other and lead to the formation of soft, chemically crosslinked protein hydrogels. We successfully used these hydrogels to encapsulate and culture human lung fibroblast cells in 3D, as well as used them to conduct drug loading and release experiments to test whether these hydrogels can be used as a controlled drug delivery vehicle.

Materials and Methods Protein Engineering Genes containing SpyCatcher and SpyTag sequences were purchased from Genscript (Piscataway, NJ, US). Subsequent genes encoding the multi-domain proteins GB1-Spycatcher (GSc), GB1-Spytag (GSt), GB1-Spytag dimer ((GSt)2), GB1-Spycatcher trimer ((GSc)3), GB1Spytag tetramer ((GSt)4), and FnIII-(GB1-Spycatcher trimer) (F(GSc)3) were constructed in the cloning vector pUC19 following a well-established stepwise construction scheme44,45. This method uses the identical sticky ends generated by BamHI and BglII restriction enzymes to build genes encoding multi-domain proteins in a stepwise fashion44,45. Then, genes for multi-domain proteins as well as that for cyan fluorescence protein (CFP) were subcloned from pUC19 vector


Page 4 of 20

Page 5 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

into the expression vector pQE80L, which carries an N-terminal His-tag sequence. Complete amino acid sequences of the constructed proteins are shown in Fig. S2. It is of note that there is an –Arg-Ser- (-RS-) linker sequence between adjacent domains, which results from restriction sites used in gene construction. The expression vector was transformed into the Escherichia coli (E. coli) strain DH5α. Bacteria culture was grown at 37 ºC in a 2.5% lysogeny broth (LB) containing 0.1 g/L ampicillin. Protein overexpression was induced with 0.5 mM isopropyl-1-βD-thiogalactoside (IPTG) when the OD600 reached ~0.8. Protein expression continued for 4 h. The bacterial cells were harvest by centrifugation at 4000 rpm for 10 min, and lysed using a 2.5 mg/mL lysozyme solution. Proteins were harvested from the soluble fraction via Co2+-affinity chromatography; GSc, GSt and (GSt)2 solutions were concentrated using Amicon Ultra Centrifugal Filter Units, and the solvent was subsequently changed to PBS. (GSc)3, (GSt)4, F(GSc)3 and CFP solutions were dialyzed against deionized water (changed every 6 hours) for 24 h to remove all salts. After dialysis, proteins were lyophilized. Molecular weights of (GSc)3, (GSt)4, F(GSc)3 proteins were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Coupling of GSc/GSt and GSc/(GSt)2 To check the reactivity of SpyCatcher and SpyTag-containing proteins in phosphate buffer saline (PBS) solution (pH7.4), 50 µM protein solutions of GSc, GSt and (GSt)2 were used. Reactions between GSc and GSt, GSc and (GSt)2 (at an Sc:St stoichiometry of 1:1)were carried out at room temperature for 1 hour. The products GSc-GSt and GSc-(GSt)2-GSc were analyzed via SDS-PAGE using a 15% polyacrylamide gel. The amount of reactants and products were quantified from the PAGE gel using ALPHAVIEW software (ProteinSimple, San Jose, CA, USA). Hydrogel construction and swelling test Purified proteins were dissolved in PBS to obtain protein solutions at desired concentrations. Corresponding Sc- and St-containing proteins solutions were mixed at a Sc:St stoichiometry of 1:1 to form hydrogels. Hydrogels were weighted after the gelation was completed (after 30 minutes of mixing, W0), and then incubated in PBS at 37 ºC. After certain


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

time, the hydrogels were weighted again after removing excess buffer (Wt). The swelling degree (SD) of the hydrogels was calculated using the following formula35 SD (%) = (Wt –W0) / W0 *100%. Rheology measurements Frequency-, strain- and time-dependent viscoelastic moduli (G’ and G”) were measured using a TA instruments Discovery Hybrid Rheometer equipped with a 8mm flat plate. The mechanical properties of (GSc)3-(GSt)4 were measured in frequency and time modes at 25 ºC with a strain of 2% and a test frequency of 10 rad/sec. The storage moduli and loss moduli were recorded over a 300 second period after the protein solutions were mixed, noting that one 5% sample was recorded for 4000 seconds, followed by a frequency-dependent sweep from 0.1 to 10 Hz. Preparation of cell-laden F(GSc)3/(GSt)4 hydrogels Human lung fibroblasts (HFL1) were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and cultured following recommended protocols. HFL1 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone). To prepare cell-laden hydrogels, purified F(GSc)3 and (GSt)4 proteins were dissolved in DMEM at desired concentrations. HFL1 cells were suspended in DMEM, where suspensions containing approximately 10,000 cells were pipetted into each Eppendorf tube and centrifuged to remove the supernatant. The cells were then re-suspended in a 40 µL (GSt)4 solution, and 60 µL of the F(GSc)3 solution added to initiate gelation. Solutions were mixed thoroughly, and then allowed to gelate for 30 minutes. The final cell concentration in each gel was 100,000 mL-1. Then the cell-laden gels were moved into a 48well plate and immersed in DMEM. Culture media were refreshed daily. Live/Dead Assay LIVE/DEAD Viability Kit for Mammalian Cells (Thermofisher Scientific) was used to ascertain the viability of cell cultures. LIVE/DEAD assay solutions for cell staining were prepared according to the manufacturer’s protocols. At certain time intervals (2 h, 24 h, 48 h and 96 h), cell laden hydrogels were rinsed with PBS for three times. Samples were then incubated in


Page 6 of 20

Page 7 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

LIVE/DEAD assay solution for 30 minutes. After staining, hydrogels were washed with PBS. Then the hydrogels were imaged using a confocal laser scanning microscope (Nikon). Images were taken at a magnification of x10 and an imaging area of 1.273 x 1.273 mm. The scanning was done in z-stack mode, and the height between each layer was 5 nm. Images reported in this study were compiled from 61 layers (300 nm total). Cyan fluorescence protein (CFP) loading and release assay CFP was loaded into hydrogels via in situ gelation. Briefly, a 10 µg/mL CFP solution was prepared by dissolving CFP in PBS (pH7.4). (GSc)3 and (GSt)4 were then dissolved in CFP solutions, followed by mixing (GSc)3/CFP and (GSt)4/CFP solutions to form CFP-loaded hydrogels. After 1 hr gelation, hydrogels were moved into 15 mL Falcon tubes containing 10 mL of PBS to test the CFP release behavior. To determine the amount of unloaded CFP, 2 mL of PBS was added to each Eppendorf tube used for preparing (GSc)3/CFP and (GSt)4/CFP solutions. To quantify the amount of released CFP at specific time points, 1 mL PBS was withdrawn from each Falcon tube and used for fluorescence measurements. Then 1 mL of fresh PBS was added into the Falcon tube to keep the PBS volume constant. The concentration of unloaded and released CFP was quantified using a fluorimeter at excitation and emission wavelengths of 420 nm and 485 nm, respectively.

Results and Discussion Engineering SpyCatcher and SpyTag-containing tandem modular proteins to construct hydrogels In order to use SpyCatcher-SpyTag chemistry to engineer protein hydrogels, we constructed two tandem modular proteins: (GB1-SpyCatcher)3 and (GB1-SpyTag)4 (Fig. S2). GB1 is a small globular protein with 56 residues that has been extensively used in our previous studies as a foundation for engineering protein-based hydrogels18,24,33. Because (GB1SpyCatcher)3 and (GB1-SpyTag)4 can be considered tri- and tetra-functional, reaction between SpyCatcher and SpyTag in these two multi-functional proteins should lead to the formation of a protein network, if the SpyCatcher-SpyTag chemistry could proceed without any difficulty in the tandem modular protein constructs.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The reaction between SpyCatcher and SpyTag will lead to the reconstitution of a folded CnaB2 domain with a typical β-barrel structure. It is not clear whether flanking the SpyTag sequence with folded globular GB1 domains sterically hinders the interaction between SpyTag and SpyCatcher. To answer this question and examine the feasibility of using SpyCatcherSpyTag chemistry to construct tandem modular protein-based hydrogels, we used GB1SpyCatcher (GSc), GB1-SpyTag (GSt), and (GB1-SpyTag)2 ((GSt)2) as model systems to examine the reactivity of SpyTag flanked with GB1. We mixed GSc with GSt at a 1:1 molar ratio, and GSc with (GSt)2 at a 2:1 ratio and allowed them to react in PBS at room temperature for 1 hour. Fig. 1 shows a picture of the SDS-PAGE gel containing the products of these two reactions (lane 4 and 5), as well as the initial reactants (lane 1-3). It is evident that after 1 hour, the three bands representing the initial reactants GSc (molecular weight (M.W.)= 20.3 kDa), GSt (M. W.= 9.2 kDa) and (GSt)2 (M. W.= 17.6 kDa) disappeared, while two new bands at ~30 kDa and ~58 kDa appeared. These new bands are in good agreement with the molecular weights of the expected products GSt-GSc (30.5 kDa) and (GSt)2GScGSc (58.2 kDa), clearly indicating that GSc reacted with GSt and (GSt)2 stoichiometrically, and that the reaction is close to completion after 1 hour. This result shows that flanking SpyTag sequence with GB1 does not hinder the reactivity of SpyCatcher-SpyTag, and that this chemistry can proceed rapidly in our tandem modular proteins at room temperature. Therefore, SpyCatcher-Spytag chemistry can be used to efficiently engineer protein-based, chemically crosslinked tandem modular hydrogels.

Figure 1. SDS-PAGE analysis showed that SpyCatcher and SpyTag can react with each other stoichiometrially. Lane 1: GB1-Sc; Lane 2: GB1-St; Lane 3: (GB1-St)2, Lane 4: the product of reacting GB1-Sc with GB1-St at a molar ratio of 1:1; and Lane 5: the product of reacting GB1-Sc with (GB1-St)2 at a molar ratio of 2:1.


Page 8 of 20

Page 9 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

SpyCatcher–SpyTag chemistry mediated hydrogel formation To construct GSc-GSt protein hydrogels, trifunctional (GSc)3 and tetrafunctional (GSt)4 proteins were overexpressed in the E. Coli. strain DH5α, and purified using Co2+ affinity chromatography. After dialysis against deionized water for 24 hours, both proteins were lyophilized. The resultant proteins have a white sponge-like solid appearance, and can be dissolved in water, PBS, and the cell culture medium DMEM. The measured molecular weights for both the purified proteins (GSc)3 and (GSt)4 are in good agreement with theoretical values, as evaluated by SDS-PAGE (Fig. S3). To construct hydrogels, (GSc)3 and (GSt)4 were dissolved in PBS at concentrations of 5%, 10% and 15%. Upon mixing (GSc)3 and (GSt)4 at a 4:3 molar ratio at room temperature, gelation occurred very rapidly (within 5 minutes), as evidenced by the loss of fluidity in the protein solution when the tube was inverted. For the 10% and 5% hydrogel, gelation occurred immediately following mixing. It is of note that both protein solutions are quite viscous, and when the concentration is 20%, the protein solution became too viscous to be pipetted. After gelation, gels were cured for 30 minutes and removed from their Eppendorf tubes. All hydrogels were self-standing, including the 5% gel, which was very fragile (Fig. 2A). After incubating for 20 hours in PBS, the hydrogels swelled slightly (Fig. 2B, and Fig. S4), and exhibited virtually no erosion, which is in sharp contrast with significant erosion observed for hydrogels physically crosslinked using GB1-based tandem modular proteins18,33. This signals the significant stability of GSc-GSt based hydrogels, deriving from the nature of chemical crosslinking initiated by the SpyCatcher-SpyTag chemistry. A schematic of the crosslinked hydrogel network is shown in Fig. 2C. To measure the mechanical properties of these hydrogels, we carried out shear rheology experiments in the time, strain and frequency-sweep modes, in which the storage (G’) and loss moduli (G”) of the hydrogel were monitored after manual mixing of two protein solutions. Fig. 3A shows the results of these experiments at a fixed strain and frequency for 5% hydrogel. The strain and frequency were chosen to be within the linear viscoelastic regime. After mixing the two protein solutions, the storage modulus increases rapidly in a nonlinear fashion. The increase of storage modulus with time can be well described by a double exponential (black curve) (Fig. S5). The initial gelation phase is fast with a half-life of 164 seconds, where the storage modulus


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 20

increase to ~60 Pa within 260 seconds. The second phase is slow with a half-life of 1760 seconds, where the storage modulus continued to increase after 1 hour. During the entire gelation process, the loss modulus G’’ remained roughly constant at ~10 Pa (Fig. S5, blue curve).

Figure 2. (GSc)3 and (GSt)4 polyproteins readily form hydrogels under ambient conditions. A) Photographs of (GSc)3/(GSt)4 hydrogels showed that these hydrogels are self-standing. These hydrogels were taken out of Eppendorf tubes after 30 minutes of gelation, and the numbers indicate the percentage of the hydrogels. B) (GSc)3/(GSt)4 hydrogels swell in PBS (up panel: at 0 h; and bottom panel: after 20 h in PBS). C) Schematic of the hydrogel formed by (GSc)3 and (GSt)4 polyproteins. The red bar indicates the isopeptide bond formed between Spycatcher and SpyTag.

Compared with the 5% hydrogel, the 10% and 15% hydrogels showed improved mechanical properties; specifically, the storage modulus of the 10% hydrogel increased to ~350


Page 11 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Pa, and that of the 15% hydrogel increased to ~600 Pa (Fig. 3A). Moreover, the storage modulus of the (GSc)3-(GSt)4 hydrogel remains largely constant from 0.01 to 20 rad/s at 25 ºC, (Fig. 3B and Fig. S6A, S6C). During the strain-sweep experiment, these hydrogels were stable up to 100% applied strain (Fig. 3C, and Fig. S6B, S6D). Taken together, these results indicate that the (GSc)3-(GSt)4-based hydrogels are covalently crosslinked, stable hydrogels.

Figure 3. Rheology characterization of the GSc)3/(GSt)4 hydrogels. A) Changes of G’ as a function of time after mixing (GSc)3 and (GSt)4 at a molar ratio of 4:3 at different concentrations (5%, 10% and 15%) at 25 ºC. It appears that 15% protein solution geled slower than the10% gels. This is likely caused by the inhomogeneity of the mixing of the 15% solution due to its high viscosity. B) Frequency sweep test of the 5% GSc)3/(GSt)4 hydrogel at 25 ºC. Both G’ and G’’ were monitored. C) Strain sweep test of the 5% GSc)3/(GSt)4 hydrogel at 25 ºC. Both G’ and G’’ were monitored.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 20

Encapsulation and 3D culture of human lung fibroblast in GSt-GSc hydrogels Due to their three-dimensional porous network structure, hydrogels have been used as artificial matrices for cell encapsulation and 3D cell culture, as well as drug carriers. Here we evaluated the efficacy of our novel GSt-GSc hydrogels towards these critical applications. Since GB1 domains are non-cell binding elastomeric proteins, cell-binding sequence/domain need to be incorporated into the protein hydrogels in order to use GSt-GSc hydrogels as an artificial matrix for 3D cell encapsulation and 3D cell culture. For this purpose, we fused the third firbronectin type III (FnIII) domain from the human extracellular matrix protein tenascin46 (TNfn3) to the N-terminus of (GSc)3 to obtain TNfn3-(GSc)3. TNfn3 was used as it contains the Arg–Gly–Asp (RGD) sequence in its folded three-dimensional structure and plays important roles in mediating cell adhesion and cell-matrix interactions24,47,48. We found that TNfn3-(GSc)3 and (GSt)4 can form hydrogels with physical and mechanical properties similar to hydrogels formed by (GSc)3 and (GSt)4. Human lung fibroblasts (HFL1) were used for cell encapsulation and 3D cell culture experiments. For this, HFL1 were suspended in F(GSc)3 solutions, and then (GSt)4 solution as added to initiate gelation. Due to the fast gelation, the cells should have been encapsulated roughly evenly in the hydrogel network after 5 minutes. After gelation proceeded for 30 minutes to ensure the complete gelation, cell-laden hydrogels were immersed in the cell culture medium DMEM. We did not observe any loss of HFL1 into the DMEM media after 24 hours of incubation, suggesting that HFL1 were successfully encapsulated in the hydrogel. Because the hydrogel contains the cell adhesive TNfn3 domain within its 3-D network, we anticipated that HFL1 could adhere to the hydrogel scaffold, and remain viable in the hydrogel scaffold. To examine the cytotoxicity of Sc- and St-containing proteins and the SpyCatcher-SpyTag chemistry, we carried out live/dead assay. We treated HFL1 cells with the Live/Dead viability stain at several time intervals (2 hours, 24 hours, 48 hours and 96 hours), and used laser scanning confocal microscope to visualize stained HFL1 cells. Fig. 4A shows stacked images encompassing an area of 1.273×1.273 mm2 and 300 nm in depth. Loaded cells exhibited high viability (>90%) within all the hydrogels during the 96 hours culturing period (Fig. 4B), indicating the low cytotoxicity and high cellular compatibility of both the protein constructs as well as the SpyCatcher-SpyTag cross-linking chemistry. It is important to note that HFL1 cells integrated into the 3-D hydrogel network largely displayed a round, rather than elongated, morphology, despite the presence of cell adhesion TNfn3 domains. It is well


Page 13 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

known that the substrate stiffness significantly impacts adhesion structures and dynamics, including cytoskeleton assembly and cell spreading49,50. Specifically, fibroblast cells tend to spread on stiffer substrates, with a Young’s modulus in the tens of kPa. It is likely that round HFL1 morphology we observed is due to the low storage modulus of our hydrogels. This is different from morphologies observed by Sun et al, who found that mouse 3T3 fibroblasts show elongated morphology when encapsulated in a 3-D hydrogel network (with a storage modulus of a few hundreds Pa) formed through SpyTag-SpyCatcher chemistry, using elastin-like proteins as building blocks28.

Figure 4. Confocal laser scanning microscopy analysis of 3D encapsulation of human fibroblast in F(GSc)3/(GSt)4 hydrogels. A) Cumulative images of cell-laden F(GSc)3/(GSt)4 hydrogels at 2h, 24h, 48h and 96h after LIVE/DEAD staining. The imaging area was 1.243×1.243 mm2 (length × width) with a depth of 300 nm. B) Viability of the fibroblast cells in gel samples.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In vitro loading and release of cyan fluorescence protein (CFP) as a model drug delivery system To examine the potential of using GSt-GSc hydrogels as drug carriers, we carried out in vitro loading and release experiments using cyan fluorescent protein (CFP) as a model system. CFP is 238 amino acid residues long, and has been widely used in live cell imaging. The intrinsic fluorescence of CFP provides a convenient way to determine the concentration of CFP unloaded and released by our model system. The encapsulation efficiency (EE) was calculated by: EE (%) = (W0 - WUL) / W0 * 100% Where WUL is the concentration of unloaded CFP (in nmol), determined by measuring the CFP remaining in solution after gelation, and W0 is the total concentration of CFP initially loaded. Taking advantage of the miscibility of CFP with both GSc/GSt protein solutions for hydrogelation, we could load CFP into hydrogel during the gelation process with encapsulation efficiency of ~70% (75± 8%, 72±1% and 71±1% for 5%, 10% and 15% hydrogels, respectively, n=3). This in situ loading process resulted in greater encapsulation efficiencies when compared with the traditional soaking-absorbing loading method. The viscosity of protein solutions containing CFP was slightly greater than the protein solution at same concentration in PBS.

Figure 5. Time course of the in vitro release of cyan fluorescence protein as a model drug within (GSc)3/(GSt)4 hydrogels in 48 hours. The inset is a zoomed view of the release profile within the first 6 hours. Experiments were carried out in triplicate and the error bars correspond to the standard deviation of the data.


Page 14 of 20

Page 15 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

CFP loaded hydrogels were prepared by mixing (GSc)3 and (GSt)4 solutions at desired molar ratios in the CFP solution. After 1 hour of gelation, hydrogels were immersed in 10 mL PBS, and samples were taken at specific time intervals to determine the cumulative release. As shown in Fig. 5, all three hydrogels quickly and continuously released CFP over the first six hours, when 87.1% (5% hydrogel), 79.9% (10% hydrogel) and 79.6% (15% hydrogel) of the loaded CFP was released. During the 6 – 48 hour time period, only another ~10% of the CFP was released. At the 48 hour point, the cumulative release ratios of the 5%, 10%, 15% hydrogels were 94.8%, 94.3% and 89.5%, respectively. After 48 hours, the release is almost negligible. Our results indicate that the release rates and behavior was not significantly different between hydrogel concentrations, despite the difference in the swelling degree of these three hydrogels. It is of note that the release experiments were carried out under the condition that the hydrogel is not degradable, and thus the release of CFP is likely a diffusion-controlled process51. The fact that the release rates and behavior were not significantly different between hydrogel concentration is likely due to the fact that the hydrogel pore size is big enough that the diffusion of CFP in the hydrogel network is essentially similar, giving rise to similar release rates.

Conclusions We demonstrated the utility of SpyCatcher-SpyTag chemistry in engineering novel tandem modular protein-based hydrogels. These resultant (GSc)2-(GSt)4 hydrogels are soft, stable and biologically compatible, and are able to both successfully encapsulate human lung fibroblast and serve as a vessel for drug delivery. Our results show that the SpyCatcher-SpyTag chemistry is robust even when folded globular domains flank the SpyTag. That sterics do not hinder the SpyTag chemistry means that the SpyCatcher-SpyTag chemistry could offer endless possibilities to construct protein hydrogels that incorporate multiple biologically important functional domains without constraining the location of these domains within tandem modular protein building blocks. Therefore, our results suggest that SpyCatcher-SpyTag chemistry can serve as an efficient, cyto-compatible means of chemically crosslinking protein hydrogels for applications in tissue engineering, drug delivery, as well as fundamental mechanobiological model system. It is important to note that crosslinking is not complete in our (GSc)3-(GSt)4 hydrogel, which is similar to the reported SpyCatcher-SpyTag-ELP hydrogels, resulting in a relatively low


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

storage modulus28. This is a potential limitation of using SpyCatcher-SpyTag engineered protein hydrogels in biological applications, noting that mechanical substrate properties, including the Young’s modulus, can provide important physical cues towards influencing cell behaviors. Therefore, developing effective means to tune the mechanical properties of SpyCatcher-SpyTag based protein hydrogels over a much broader range (from 10s Pa to 10s kPa) will be critical in order to fully explore the potential of SpyCatcher-SpyTag hydrogels. Towards this goal, understanding the factors that influence the SpyCatcher-SpyTag reaction in a highly concentrated protein solution and subsequently identifying practical ways to tune the crosslinking degree of the hydrogels are of critical importance. Experimental efforts are currently underway to address this important challenge.

Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Schematics of the SpyCatcher-SpyTag chemistry; Sequences of the constructed elastomeric proteins; SDS-PAGE analysis of the proteins; Swelling test of the constructed SpyCatcher-SpyTag hydrogels; and Rheology characterization of the protein hydrogels.

Acknowledgements This work is supported by Canadian Institutes of Health Research, Canada Foundation for Innovation and Canada Research Chairs program.


Page 16 of 20

Page 17 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

References (1) Langer, R.; Tirrell, D. A. Designing materials for biology and medicine. Nature 2004, 428, 487-492. (2) Lee, K. Y.; Mooney, D. J. Hydrogels for tissue engineering. Chem. Rev. 2001, 101, 1869-1879. (3) Ren, K.; He, C.; Xiao, C.; Li, G.; Chen, X. Injectable glycopolypeptide hydrogels as biomimetic scaffolds for cartilage tissue engineering. Biomaterials 2015, 51, 238-249. (4) Lv, S. S.; Bu, T. J.; Kayser, J.; Bausch, A.; Li, H. B. Towards constructing extracellular matrix-mimetic hydrogels: An elastic hydrogel constructed from tandem modular proteins containing tenascin FnIII domains. Acta Biomater. 2013, 9, 6481-6491. (5) Pritchard, E. M.; Kaplan, D. L. Silk fibroin biomaterials for controlled release drug delivery. Exp. Opin. Drug Deli. 2011, 8, 797-811. (6) MacEwan, S. R.; Chilkoti, A. Elastin-Like Polypeptides: Biomedical Applications of Tunable Biopolymers. Biopolymers 2010, 94, 60-77. (7) Kopecek, J. Hydrogel biomaterials: A smart future? Biomaterials 2007, 28, 51855192. (8) Kopecek, J.; Yang, J. Smart self-assembled hybrid hydrogel biomaterials. Angew Chem Int Ed Engl 2012, 51, 7396-7417. (9) Lutolf, M. P.; Gilbert, P. M.; Blau, H. M. Designing materials to direct stem-cell fate. Nature 2009, 462, 433-441. (10) Lutolf, M. P.; Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotech. 2005, 23, 47-55. (11) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Reversible hydrogels from self-assembling artificial proteins. Science 1998, 281, 389-392. (12) Parisi-Amon A, H., SC. In Biomedical Engineering Handbook, 4th Edition; Bronzino JD, P. D., Ed.; CRC Press, Taylor & Francis Group: Boca Raton, FL, 2012; Vol. 4. (13) Banta, S.; Wheeldon, I. R.; Blenner, M. In Annual Review of Biomedical Engineering, Vol 12; Yarmush, M. L., Duncan, J. S., Gray, M. L., Eds. 2010; Vol. 12, p 167-186. (14) Li, H.; Kong, N.; Laver, B.; Liu, J. Hydrogels Constructed from Engineered Proteins. Small 2016, 12, 973-987. (15) Wang, C.; Stewart, R. J.; Kopecek, J. Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains. Nature 1999, 397, 417-420. (16) DiMarco, R. L.; Heilshorn, S. C. Multifunctional Materials through Modular Protein Engineering. Adv. Mater. 2012, 24, 3923-3940. (17) Foo, C.; Lee, J. S.; Mulyasasmita, W.; Parisi-Amon, A.; Heilshorn, S. C. Twocomponent protein-engineered physical hydrogels for cell encapsulation. Proc. Natl. Acad. Sci. USA 2009, 106, 22067-22072. (18) Cao, Y.; Li, H. Engineering tandem modular protein based reversible hydrogels. Chem. Comm. 2008, 4144-4146. (19) Shen, W.; Zhang, K. C.; Kornfield, J. A.; Tirrell, D. A. Tuning the erosion rate of artificial protein hydrogels through control of network topology. Nat. Mater. 2006, 5, 153-158. (20) Kong, N.; Li, H. Protein Fragment Reconstitution as a Driving Force for SelfAssembling Reversible Protein Hydrogels. Adv. Funct. Mater. 2015, 25, 5593-5601. (21) Xiao, W.; He, J.; Nichol, J. W.; Wang, L.; Hutson, C. B.; Wang, B.; Du, Y.; Fan, H.; Khademhosseini, A. Synthesis and characterization of photocrosslinkable gelatin and silk fibroin interpenetrating polymer network hydrogels. Acta Biomater. 2011, 7, 2384-2393.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(22) Elvin, C. M.; Carr, A. G.; Huson, M. G.; Maxwell, J. M.; Pearson, R. D.; Vuocolo, T.; Liyou, N. E.; Wong, D. C. C.; Merritt, D. J.; Dixon, N. E. Synthesis and properties of crosslinked recombinant pro-resilin. Nature 2005, 437, 999-1002. (23) Lim, D. W.; Nettles, D. L.; Setton, L. A.; Chilkoti, A. In situ cross-linkinig of elastin-like polypeptide block copolymers for tissue repair. Biomacromolecules 2008, 9, 222230. (24) Lv, S.; Dudek, D. M.; Cao, Y.; Balamurali, M. M.; Gosline, J.; Li, H. Designed biomaterials to mimic the mechanical properties of muscles. Nature 2010, 465, 69-73. (25) Charati, M. B.; Ifkovits, J. L.; Burdick, J. A.; Linhardt, J. G.; Kiick, K. L. Hydrophilic elastomeric biomaterials based on resilin-like polypeptides. Soft Matter 2009, 5, 3412-3416. (26) McGann, C. L.; Levenson, E. A.; Kiick, K. L. Resilin-Based Hybrid Hydrogels for Cardiovascular Tissue Engineering. Macromol. Chem. Phys. 2013, 214, 203-213. (27) Chung, C.; Lampe, K. J.; Heilshorn, S. C. Tetrakis(hydroxymethyl) Phosphonium Chloride as a Covalent Cross-Linking Agent for Cell Encapsulation within Protein-Based Hydrogels. Biomacromolecules 2012, 13, 3912-3916. (28) Sun, F.; Zhang, W.-B.; Mahdavi, A.; Arnold, F. H.; Tirrell, D. A. Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry. Proc. Natl. Acad. Sci. USA 2014, 111, 11269-11274. (29) Tatham, A. S.; Shewry, P. R. Elastomeric proteins: biological roles, structures and mechanisms. Trends Biochem. Sci. 2000, 25, 567-571. (30) Fong, E.; Tirrell, D. A. Collective Cell Migration on Artificial Extracellular Matrix Proteins Containing Full-Length Fibronectin Domains. Adv. Mater. 2010, 22, 5271-5275. (31) Wheeldon, I. R.; Barton, S. C.; Banta, S. Bioactive proteinaceous hydrogels from designed bifunctional building blocks. Biomacromolecules 2007, 8, 2990-2994. (32) Fang, J.; Mehlich, A.; Koga, N.; Huang, J. Q.; Koga, R.; Gao, X. Y.; Hu, C. G.; Jin, C.; Rief, M.; Kast, J.; Baker, D.; Li, H. B. Forced protein unfolding leads to highly elastic and tough protein hydrogels. Nature Comm. 2013, 4, 10. (33) Lv, S. S.; Cao, Y.; Li, H. B. Tandem Modular Protein-Based Hydrogels Constructed Using a Novel Two-Component Approach. Langmuir 2012, 28, 2269-2274. (34) Fancy, D. A.; Kodadek, T. Chemistry for the analysis of protein-protein interactions: Rapid and efficient cross-linking triggered by long wavelength light. Proc. Natl. Acad. Sci. USA 1999, 96, 6020-6024. (35) Elvin, C. M.; Vuocolo, T.; Brownlee, A. G.; Sando, L.; Huson, M. G.; Liyou, N. E.; Stockwell, P. R.; Lyons, R. E.; Kim, M.; Edwards, G. A.; Johnson, G.; McFarland, G. A.; Ramshaw, J. A. M.; Werkmeister, J. A. A highly elastic tissue sealant based on photopolymerised gelatin. Biomaterials 2010, 31, 8323-8331. (36) Fang, J.; Li, H. B. A Facile Way to Tune Mechanical Properties of Artificial Elastomeric Proteins-Based Hydrogels. Langmuir 2012, 28, 8260-8265. (37) Elvin, C. M.; Brownlee, A. G.; Huson, M. G.; Tebb, T. A.; Kim, M.; Lyons, R. E.; Vuocolo, T.; Liyou, N. E.; Hughes, T. C.; Ramshaw, J. A. M.; Werkmeister, J. A. The development of photochemically crosslinked native fibrinogen as a rapidly formed and mechanically strong surgical tissue sealant. Biomaterials 2009, 30, 2059-2065. (38) Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy, V. T.; Howarth, M. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl. Acad. Sci. USA 2012, 109, E690-E697.


Page 18 of 20

Page 19 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(39) Zakeri, B.; Howarth, M. Spontaneous Intermolecular Amide Bond Formation between Side Chains for Irreversible Peptide Targeting. J. Amer. Chem. Soc. 2010, 132, 4526-+. (40) Kang, H. J.; Coulibaly, F.; Clow, F.; Proft, T.; Baker, E. N. Stabilizing isopeptide bonds revealed in Gram-positive bacterial pilus structure. Science 2007, 318, 1625-1628. (41) Li, L.; Fierer, J. O.; Rapoport, T. A.; Howarth, M. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. J. Mol. Biol. 2014, 426, 309-317. (42) Reddington, S. C.; Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr. Opin. Chem. Biol. 2015, 29, 94-99. (43) Zhang, W.-B.; Sun, F.; Tirrell, D. A.; Arnold, F. H. Controlling Macromolecular Topology with Genetically Encoded SpyTag-SpyCatcher Chemistry. J. Amer. Chem. Soc. 2013, 135, 13988-13997. (44) Cao, Y.; Lam, C.; Wang, M. J.; Li, H. B. Nonmechanical protein can have significant mechanical stability. Angew. Chem. Int. Ed. 2006, 45, 642-645. (45) Carrion-Vazquez, M.; Oberhauser, A. F.; Fowler, S. B.; Marszalek, P. E.; Broedel, S. E.; Clarke, J.; Fernandez, J. M. Mechanical and chemical unfolding of a single protein: A comparison. Proc. Natl. Acad. Sci. USA 1999, 96, 3694-3699. (46) Leahy, D. J.; Hendrickson, W. A.; Aukhil, I.; Erickson, H. P. Structure of a Fibronectin Type-Iii Domain from Tenascin Phased by Mad Analysis of the Selenomethionyl Protein. Science 1992, 258, 987-991. (47) Jones, P. L.; Jones, F. S. Tenascin-C in development and disease: gene regulation and cell function. Matrix Biol 2000, 19, 581-596. (48) Yokosaki, Y.; Matsuura, N.; Higashiyama, S.; Murakami, I.; Obara, M.; Yamakido, M.; Shigeto, N.; Chen, J.; Sheppard, D. Identification of the ligand binding site for the integrin alpha9 beta1 in the third fibronectin type III repeat of tenascin-C. J Biol Chem 1998, 273, 11423-11428. (49) Discher, D. E.; Janmey, P.; Wang, Y. L. Tissue cells feel and respond to the stiffness of their substrate. Science 2005, 310, 1139-1143. (50) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 2006, 126, 677-689. (51) Fu, Y.; Kao, W. J. Drug release kinetics and transport mechanisms of nondegradable and degradable polymeric delivery systems. Expert Opin Drug Deliv 2010, 7, 429444.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC Graphic


Page 20 of 20

Engineering Protein Hydrogels Using SpyCatcher-SpyTag Chemistry

Recommend Documents