Name: synthesis of an intein-mediated artificial protein hydrogel
Uploaded: 2014-01-27T13:00:00
Description: Watch this Scientific Journal Video about Synthesis of an Intein-mediated Artificial Protein Hydrogel at JoVE.com

The authors would like to acknowledge Dr. David Tirrell (Caltech) for his kind gift of the plasmid pQE9 AC10Atrp12, Dr. Tom Muir (Princeton University) for his kind gift of the plasmid KanR-IntRBS-NpuNC-CFN11, Dr. Takehisa Matsuda (Kanazawa Institute of Technology, Hakusan, Ishikawa, Japan) for his kind gift of the plasmid pET30-CutA-Tip110, and Dr. Jay D. Keasling (UC Berkley) for his kind gift of the plasmid pJD75713. This work was supported in part by the National Science Foundation CAREER, US Air force YIP and Norman Hackman Advanced Research Program.

1. Plasmid Construction
NOTE: All genes were amplified under standard PCR reactions using Phusion High-Fidelity DNA Polymerase per the manufacturer&#39;s specifications. Primers used for cloning have been described previously9. All constructs are listed in Table 1.
<ol>
	<li>To generate CutA-NpuN (N, Table 1):
	<ol>
		<li>PCR amplify CutA and NpuN genes from plasmids pET30-CutA-Tip110 and KanR-IntRBS-NpuNC-CFN11</s...

<ol>
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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...

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 intein2,3 and a small trimeric protein (12 kDa) CutA from Pyrococcus horikoshii was used as the crosslinker protein4,5. 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 (t1/2 = 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 &#176;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 &#34;docking station peptide&#34; (DSP) is incorporated into one of the hydrogel building blocks to facilitate stable immobilization of a &#34;docking protein&#34; (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 &#176;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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			Comments
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			Phusion High Fidelity DNA polymerase
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			New England BioLabs
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			M0530S
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			Competent Escherichia coli BL21 (DE3)
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			New England BioLabs
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			C2527I
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			Luria Bertani
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			VWR
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			90003-350
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			Bacto Agar Media
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			VWR
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			<td nowrap="nowrap" style="width:118px;height:21px;">
			<a href="https://us.vwr.com/store/catalog/product.jsp?catalog_number=90000-760">90000-760</a>
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			kanamycin sulfate
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			VWR
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			<td nowrap="nowrap" style="width:118px;height:21px;">
			<a href="https://www.vwrsp.com/catalog/product/index.cgi?catalog_number=97061-602">97061-602</a>
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			IPTG
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			VWR
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			EM-5820
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			Imidazole
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			VWR
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			EM-5720
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			Urea
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			VWR
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			EM-9510
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			Dithiothreitol (DTT)
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			Fisher
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			BP172-5
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			Protease Inhibitor cocktail
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			Roche Applied Science
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			11836153001
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			DPBS
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			VWR
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			82020-066
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			Brilliant Blue R
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			Acros Organics
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			A0297990
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			Sodium Azide
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			Fisher
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			AC190380050
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			<td nowrap="nowrap" style="width:137px;height:21px;">
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			Horseradish peroxidase
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			Sigma
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			P8125-5KU
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			N,N-dimethyl-p-phenylene diamine
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			Fisher
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			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC408460250
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			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
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			phenol
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			Fisher
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			AC149340500
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			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
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			tert-butyl hydroperoxide
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			Fisher
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			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC180340050
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			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
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			n-heptane
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			Acros Organics
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			120340010
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			Max Q 6000
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			Misonix 200
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			Ultrafiltration Tubes
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			Amicon Ultra
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			UFC903024
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			&#160;Ni Sepharose High Performance HisTrap column
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			GE Healthcare Life Sciences
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			17-5248-01
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			HiTrap SP Sepharose FF ion exchange column
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			GE Healthcare Life Sciences
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			Molecular Devices
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			SpectraMax Gemini EM
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			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
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synthesis of an intein-mediated artificial protein hydrogel

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 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-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.

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 horikoshii4,5. Mixing of purified N and C in the presence of the reducing agent DTT induces the formation...

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Synthesis of an Intein-mediated Artificial Protein Hydrogel

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.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

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Bioengineering

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