Synthesis of an Intein-mediated Artificial Protein Hydrogel

Summary

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

Abstract

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 °C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a "docking station peptide" into the hydrogel building block enables convenient incorporation of "docking protein"-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

Introduction

Hydrogels made entirely of proteins carry the potential to significantly advance fields as diverse as tissue engineering, drug delivery and biofabrication¹. They offer advantages over traditional synthetic polymer hydrogels including biocompatibility and the potential to noninvasively support the incorporation of bioactive globular proteins.

In this work, we describe the development of a novel protein hydrogel formed via a split-intein-mediated protein trans-splicing reaction and its application as a protein immobilization scaffold (Figure 1). The building blocks for this hydrogel are two protein block-copolymers each comprising the N- or C-terminal fragment of a split intein (IN and IC) and a subunit of a multimeric crosslinker protein. The DnaE intein from Nostoc punctiforme (Npu) was used as the split intein^2,3 and a small trimeric protein (12 kDa) CutA from Pyrococcus horikoshii was used as the crosslinker protein^4,5. Different crosslinkers are joined through intein catalyzed trans-splicing reaction, leading to the formation of a highly crosslinked protein network (hydrogel). Npu intein was chosen because of its fast reaction kinetics (t_1/2 = 63 sec) and high trans-splicing yield (close to 80%)^2,3. The CutA protein was chosen as the crosslinker due to its high stability. CutA trimers have a denaturation temperature of near 150 °C and retain trimeric quaternary structure in solutions containing as much as 5 M guanidine hydrochloride ^4,6. Since subunit exchange between different crosslinkers is a major contributor of the physical hydrogel surface erosion⁷, the very strong inter subunit interaction in CutA should discourage such subunit exchanges, leading to a more stable hydrogel. One of these building blocks also contains a highly hydrophilic peptide S-fragment as the mid-block to facilitate water retention⁸.

Mixing of the two hydrogel building blocks initiates a trans-splicing reaction between the IN and IC intein fragments, generating a longer polypeptide chain with crosslinkers at both terminals. Crosslinkers from multiple such molecular units interact with each other, forming a highly crosslinked hydrogel network (Figure 1A). A specific "docking station peptide" (DSP) is incorporated into one of the hydrogel building blocks to facilitate stable immobilization of a "docking protein" (DP)-tagged target protein into the hydrogel. The use of a split intein to mediate the hydrogel assembly not only provides additional flexibility for protein hydrogel synthesis, but also enables high-density, uniform loading of the target protein throughout the entire hydrogel, as the target proteins are loaded prior to hydrogel formation.

The intein-mediated protein hydrogel is highly stable in aqueous solution with little-to-no detectable erosion after 3 months at room temperature. Stability is retained in a wide range of pHs (6-10) and temperatures (4-50 °C), and the hydrogel is also compatible with organic solvents. This hydrogel is used for the immobilization of two globular proteins: the green fluorescent protein (GFP) and the horseradish peroxidase (HRP). Hydrogel entrapping the latter protein is used to perform biocatalysis in an organic solvent.

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Protocol

1. Plasmid Construction

NOTE: All genes were amplified under standard PCR reactions using Phusion High-Fidelity DNA Polymerase per the manufacturer's specifications. Primers used for cloning have been described previously⁹. All constructs are listed in Table 1.

1. To generate CutA-NpuN (N, Table 1):
   1. PCR amplify CutA and NpuN genes from plasmids pET30-CutA-Tip1¹⁰ and KanR-IntRBS-NpuNC-CFN¹¹, respectively, using the appropriate primers.
   2. Digest these fragments with the appropriate restriction enzymes and sequentially insert these fragments into the pET26b vector between the T7 promoter and a C-terminal 6xHistidine tag to generate N (Figure 2A).
2. To generate NpuC-S-CutA (C, Table 1):
   1. PCR amplify NpuC, CutA and S fragment [AG₃(PEG)]₁₀ from plasmid KanR-IntRBS-NpuNC-CFN¹¹, pET30-CutA-Tip1¹⁰ and pQE9 AC₁₀Atrp¹², respectively, using the appropriate primers.
   2. Digest these fragments with the appropriate restriction enzymes and sequentially insert these fragments into the pET26b vector between T7 promoter and a C-terminal 6xHistidine to generate C (Figure 2B).
3. To generate NpuC-S-SH3_lig-CutA (C-SH3_lig, Table 1):
   1. PCR amplify CutA using primers containing a SH3_lig (PPPALPPKRRR) and a flexible linker (GGGGS)₂ to generate fragment SH3_lig-CutA.
   2. Replace the CutA gene from C with fragment SH3_lig-CutA.
4. To generate SH3-GFP (Table 1):
   1. Amplify the SH3 gene from plasmid pJD757¹³ using the appropriate primers.
   2. Fuse this fragment to the GFP gene and insert it into the pET26b vector between the T7 promoter (Figure 2C) and a C-terminal 6xHistidine tag.

2. Protein Expression

1. Transform 50 μl of chemically competent Escherichia coli BL21(DE3) with the appropriate expression plasmid.
2. After transformation, serially dilute these cells, and plate them on Luria-Bertani (LB)/agar plates containing 50 μg/ml kanamycin.
3. Incubate plates containing transformed cells at 37 °C for ~15 hr.
4. After incubation, pick a plate that contains 50-100 colonies and resuspend all colonies in 5 ml of LB broth.
5. Transfer suspension to 1 L LB broth containing kanamycin (50 μg/ml) and grow cells at 37 °C with shaking at 250 rpm. Monitor the absorbance at 600 nm (OD₆₀₀). Grow culture until OD₆₀₀ ~0.8.
   1. For C and C-SH3_lig, induce protein expression by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture (1 mM final concentration) and incubate the culture at 37 °C for 4 hr while shaking at 250 rpm.
   2. For N and SH3-GFP, cool the culture to ~18 °C by immersing the culture flask in an ice water bath for ~5 min. Induce protein expression by adding IPTG to the culture (1mM final concentration) and incubate the culture at 18 °C for 14-18 hr while shaking at 250 rpm.
6. After protein expression, centrifuge the culture at 6,000 x g for 20 min at 4 °C to collect the pellet. Store cell pellet at -80 °C until use.

3. Protein Purification

1. Purification of N (denaturing conditions)
   1. Resuspend cell pellets in Buffer A (Table 2) at 10 ml/g of wet pellet.
   2. Immerse the pellet suspension in an ice-water bath and disrupt cells by sonication (Amp 10, with 1 sec pulse and 6 sec pause for 1 min).
   3. Centrifuge the lysate at 16,000 x g for 20 min at 4 °C.
   4. Discard the supernatant. Resuspend the pellet in Buffer DA (containing 8 M urea) and centrifuge the suspension at 16,000 x g for 20 min at 4 °C.
   5. Pass the supernatant through a 5 ml Ni-nitrilotriacetic acid (NTA) column previously equilibrated with buffer DA.
   6. Wash column with 30 ml of Buffer DA supplemented with 45 mM imidazole. Elute purified protein using 20 ml of Buffer DA supplemented with 150 mM imidazole.
   7. Reduce the urea concentration in the protein sample to <1 mM by either one of the following methods given in 3.1.7.1 or 3.1.7.2:
      1. Dialyze protein in DPBS buffer (Table 2) at 4 °C overnight using tubes with <20 kDa cutoff.
      2. Centrifuge purified protein in a 30 kDa ultra-filtration spin column at 2,800 x g, 4 °C until the volume is less than 1 ml. Add 14 ml DPBS buffer to the column to dilute the protein sample. Repeat the centrifugation/dilution steps three more times.
   8. After buffer exchange, add dithiothreitol (DTT) to the purified protein (final 2 mM) and concentrate protein to ~100 mg/ml by centrifugation through a 30 kDa ultra-filtration spin column at 2,800 x g, 4 °C.
   9. Aliquot the concentrated protein and store at -80 °C until use.
2. Purification of C and C-SH3_lig (native condition)
   1. Resuspend cell pellets in Buffer B (pH 6.0) (Table 2) supplemented with 1x protease inhibitor cocktail at 10 ml/g of wet pellet. Use acidic buffer to minimize proteolytic degradation of the target protein.
   2. Disrupt cell suspension by sonication as described in 3.1.2. Centrifuge the lysate at 16,000 x g for 20 min at 4 °C and keep the supernatant.
   3. Pass the soluble lysate through a 5-ml Ni-NTA column previously equilibrated with buffer B.
   4. Wash column with Buffer B supplemented with 45 mM imidazole, and elute the target protein in 20 ml of Buffer B supplemented with 150 mM imidazole.
   5. For C, skip to step 3.2.6. For C-SH3_lig, carry out an additional ion-exchange purification step to remove partially degraded protein as given in steps 3.2.5.1 to 3.2.5.3
      1. Reduce NaCl concentration in C-SH3_lig to <1 mM following the procedure described in 3.1.7.
      2. Load the target protein onto a 5 ml anion exchanger beaded agarose matrix column previously equilibrated with sodium phosphate buffer (50 mM, pH 7.0).
      3. Elute target protein from the column by running a gradient from a solution containing 10 mM Tris-HCl pH 8.0 buffer to a solution containing the same buffer supplemented with 1 M NaCl. Take samples during protein elution and pool samples with the highest purity based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
   6. Buffer exchange the purified protein into DPBS buffer, as described in 3.1.7.
   7. Add DTT to the purified protein (final concentration 2 mM) and concentrate the protein to ~100 mg/ml using a 30 kDa ultra-filtration spin column as described in 3.1.8. Aliquot and store concentrated protein at -80 °C until use.
3. Purification of SH3-GFP
   1. Resuspend cell pellets using Buffer A at 10 ml/g of wet pellet.
   2. Disrupt pellet suspension by sonication as described in 3.1.2.
   3. Centrifuge the lysate at 16,000 x g for 20 min at 4 °C and collect the supernatant.
   4. Pass the supernatant (soluble lysate) through a 5-ml Ni-NTA column previously equilibrated with Buffer A.
   5. Wash column with 30 ml of Buffer A supplemented with 45 mM imidazole. Elute purified protein using 20 ml of Buffer A supplemented with 150 mM imidazole.
   6. Buffer exchange the purified protein into DPBS buffer using an approach similar to that described in 3.1.7 and concentrate the protein to ~150 mg/ml using a 30 kDa ultra-filtration spin column as described in 3.1.8.
   7. Aliquot and store purified protein at -80 °C until use.
4. SDS-PAGE analysis of purified samples containing CutA
   1. Dilute each purified protein in double-distilled water to reduce the concentration of NaCl to ~1 mM. At this NaCl concentration, most of the CutA trimer proteins run as monomers on the SDS-PAGE gels.
   2. Mix samples with 2x SDS sample buffer (0.5 M Tris-HCl, pH 6.8, 20% Glycerol, 10% w/v SDS, 0.1 % w/v bromo-phenol blue, 2% β-mercaptoethanol), incubated at 95 °C for 5 min.
   3. Load the samples onto a 12% SDS-PAGE gel. Carry out electrophoresis at a constant voltage of 200 V for ~50 min.
   4. Observe protein in the gels by staining with Coomassie brilliant blue R250 following the standard protocols (Figure 1C).

4. Hydrogel Formation

NOTE: the sample hydrogels made in this study contain 1.6 mM of each hydrogel building block unless noted otherwise. This protein concentration yields a soft and stable hydrogel. CAUTION: Sodium azide (NaN₃) is added to the hydrogel to a final concentration of 0.5% w/v to prevent bacterial contamination. NaN₃ is highly toxic and must be handled with extreme care as indicated in the Material Safety Data Sheet.

1. Calculate the volume for each of the concentrated proteins needed to achieve a final concentration of 1.6 mM in a 100 μl sample hydrogel. For example:
   Concentration of N: 100 mg/ml
   Molecular weight of N: 26.3 kDa (refer to Table 1)
   Desired Volume: 100 μl Desired Concentration: 1.6 mM
   
2. To make a 100 μl hydrogel (1.6 mM), mix C (x μl, volume calculated according to 4.1) with 5% NaN₃ (10 μl), 100 mM DTT (5 μl) and N (y μl, volume calculated according to 4.1) inside a 2 ml glass vial.
3. Add DPBS buffer ((85 - x - y) μl) to the vial to achieve a final volume of 100 μl, and manually mix all the components via a swirling motion using a pipette tip. Note: The solution becomes very viscous upon mixing.
4. Centrifuge the mixture for 2 min at 8,000 x g to remove the air bubbles.
5. Incubate the mixture at room temperature overnight to allow the intein trans-splicing reaction to reach completion. Confirm hydrogel formation by turning tube upside down. The proteins will not flow if a hydrogel is formed.
6. Estimate the intein trans-splicing yield by checking samples (0.5 μl each) collected before step 4.2 and after step 4.5 on a SDS-PAGE gel, as described in 3.4 (Figure 1C).

5. Immobilization of GFP via Docking Protein (DP) and Docking Station Peptide (DSP) Interaction

1. To make a 50 μl GFP-functionalized hydrogel (1.2 mM), combine C-SH3_lig (x μl, calculated according to 4.1) and SH3-GFP (y μl, calculated according to 4.1) at 1:1 molar ratio in a 1.7 ml microcentrifuge tube and incubate the mixture at room temperature for 30 min.
2. Add 5% NaN₃ (5 μl), 100 mM DTT (2.5 μl), (42.5 - x - y) μl DPBS to the same tube. Add N (y μl, calculated according to 4.1) to achieve a 1:1 molar ratio of N and C-SH3_lig. Mix the sample by using a pipette tip by a swirling motion.
3. Centrifuge the mixture at 8,000 x g for 2 min and incubate the mixture at room temperature overnight in the dark. A hydrogel encapsulating SH3-GFP forms during incubation.

6. Use of 1.6 mM Hydrogel as an Immobilization Scaffold for Enzymatic Reaction in Organic Solvent

1. Use the HRP as a model enzyme. Prepare a stock solution of HRP (28 mg/ml or 0.63 mM) in DPBS.
2. To make a 30 μl hydrogel (1.6 mM) entrapping HRP, combine C (x μl, calculated according to 4.1) with HRP (2 μl), 5% NaN₃ (3 μl) and DTT (1.5 μl of 100 mM) inside a 1.7 ml centrifuge tube.
3. Add N (y μl, calculated according to 4.1) and DPBS (23.5 - x - y) μl. Mix with a pipette tip with a swirling motion.
4. Centrifuge the mixture at 8,000 x g for 2 min and incubate at room temperature overnight.
   CAUTION: the regents used for the following activity assay are highly toxic. Use specific safety recommendations by the corresponding Material Safety Data Sheets.
5. For enzymatic reaction, submerge the hydrogel in 1 ml of reaction cocktail containing N,N-dimethyl-p-phenylene diamine (5.8 mM), phenol (5.8 mM) and tert-butyl hydroperoxide (2.9 mM) in n-heptane¹⁴. Manually disrupt the gel using a pipette tip to increase the contact surface area of the hydrogel and the solvent.
6. Detect HRP product, an indophenol-type dye, by measuring the optical absorbance of samples taken at different times at 546 nm in a plate reader (Figure 5).

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Results

A schematic for intein-mediated protein hydrogel formation is presented in Figure 1A. The building blocks of the hydrogel are the protein copolymers CutA-NpuN (N) and NpuC-S-CutA(C) (Figure 1A, Table 1). NpuN/C are the N-/C-fragments of the naturally split DnaE intein from Nostoc punctiforme (Npu). CutA is a stable trimeric protein from Pyrococcus horikoshii^4,5. Mixing of purified N and C in the presence of the reducing agent DTT induces the formation...

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Discussion

In this work, we demonstrated the synthesis of a highly stable intein-mediated protein hydrogel. The use of a split intein enables the hydrogel to be conditionally formed in response to the mixing of two liquid-phase components. Specifically, the split intein covalently links two liquid-phase building blocks via a trans-splicing reaction, yielding a polypeptide unit flanked by crosslinking units that in turn self assembles into a hydrogel. The mixing-induced formation of the hydrogel bypasses technical difficult...

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Materials

Name	Company	Catalog Number	Comments
Phusion High Fidelity DNA polymerase	New England BioLabs	M0530S
Competent Escherichia coli BL21 (DE3)	New England BioLabs	C2527I
Luria Bertani	VWR	90003-350
Bacto Agar Media	VWR
Kanamycin sulfate	VWR
IPTG	VWR	EM-5820
Imidazole	VWR	EM-5720
Urea	VWR	EM-9510
Dithiothreitol (DTT)	Fisher	BP172-5
Protease Inhibitor cocktail	Roche Applied Science	11836153001
DPBS	VWR	82020-066
Brilliant Blue R	Acros Organics	A0297990
Sodium Azide	Fisher	AC190380050	Caution, highly toxic
Horseradish peroxidase	Sigma	P8125-5KU
N,N-dimethyl-p-phenylene diamine	Fisher	AC408460250	Caution, highly toxic
phenol	Fisher	AC149340500	Caution, highly toxic
tert-butyl hydroperoxide	Fisher	AC180340050	Caution, highly toxic
n-heptane	Acros Organics	120340010
[header]
Shaker/Incubator	Fisher Scientific	Max Q 6000
Centrifuge	Sorvall	RC 6
Sonicator	QSonica	Misonix 200
Ultrafiltration Tubes	Amicon Ultra	UFC903024
Ni Sepharose High Performance HisTrap column	GE Healthcare Life Sciences	17-5248-01
HiTrap SP Sepharose FF ion exchange column	GE Healthcare Life Sciences	17-5156-01
Plate reader	Molecular Devices	SpectraMax Gemini EM