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

作者要感谢大卫·蒂雷尔博士（加州理工学院）的盛情质粒的礼物pQE9交流10 ATRP 12，汤姆·缪尔博士（普林斯顿大学）的盛情质粒KanR表- IntRBS-NpuNC-CFN 11的礼物，松田武久博士（技术，白山市，石川，日本金泽大学）的盛情质粒PET30-CUTA -提示1 10，博士和杰伊·D·基斯林（加州大学伯克利）的盛情质粒的礼物pJD757 13的礼物。这项工作是由美国国家科学基金会职业，美国空军叶和诺曼·哈克曼高级研究计划中的一部分支持。

1。质粒的构建注意：所有的基因都在使用每制造商的规格的Phusion高保真DNA聚合酶的标准PCR反应扩增。用于克隆的引物已经预先9所述。所有结构都列在表1中 。 <ol><li>要生成CUTA-NpuN（N，表1）： <ol><li> PCR扩增来自质粒PET30-CUTA-提示1 10和KanR表-IntRBS-NpuNC-CFN 11，分别CUTA和NpuN基因，使用适当的引物。 </li><li>消化这些片段与适当的...

<ol>
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在这项工作中，我们展示了一个高度稳定的内含肽介导的蛋白水凝胶的合成。使用一个分裂内蛋白使得能够有条件地形成响应于两个液相成分混合的水凝胶。具体而言，分割内含肽共价连接两个液相积木通过转拼反应，得到交联单元，反过来自我组装成水凝胶两侧的多肽单元。混合诱导形成水凝胶的旁路中的单组分蛋白质凝胶，其中的色谱纯化柱的凝胶介导的堵塞可能发生的合成技术困难?...

蛋白质完全由水凝胶携带可能显著推进领域等不同的组织工程，药物输送和biofabrication 1。它们比传统的合成聚合物水凝胶的优点，包括生物相容性和非侵入性地支持生物活性的球状蛋白的结合的可能性。 在这项工作中，我们描述了通过拆分内含肽介导的蛋白转拼反应及其应用作为蛋白质固定支架（ 图1）形成一种新的蛋白质凝胶的发展。此水凝胶的积木两种蛋白嵌段共聚物各自包含一个分裂的内含肽（IN和IC）和多聚体交联蛋白质的亚单位的N-或C-末端片段。该DnaE内含肽自念珠藻punctiforme（NPU）用作分割内含肽2,3和一个小的三聚体蛋白（12 kDa）的来自激烈热 CUTA horikoshii &lt;/ em&gt;的用作交联剂蛋白4,5。不同的交联剂，通过内含肽催化的转拼反应结合，导致了高交联的蛋白网络（水凝胶）的形成。 NPU内含肽的选择，因为它的快速反应动力学（T 1/2 = 63秒）和高反式剪接率（接近80％）2,3。该CUTA蛋白被选为由于其高稳定性的交联剂。 CUTA三聚物具有接近150℃的变性温度，并保持三聚体四级结构中含有多达5个M盐酸胍4,6的解决方案。由于不同的交联剂亚基之间交换的物理水凝胶表面糜烂7的主要贡献者，在CUTA很强的亚基间相互作用应该阻止这种亚基的交流，从而导致更稳定的水凝胶。一这些积木还含有高度亲水的肽的S-片段作为中间嵌段，以促进水保留8。 两种水凝胶积木混合发起内含肽片段的IN和集成电路之间的转拼反应，生成一个较长的多肽链中与交联剂在两个终端。由多个这样的分子单元的交联剂彼此相互作用，从而形成高度交联的水凝胶网络（ 图1A）。一个具体的“基座肽”（DSP）掺入水凝胶的基石之一，以促进一个“对接蛋白”（DP）-标记的目标蛋白质为水凝胶的稳定固定。使用一个分裂内含肽介导的水凝胶组件不仅提供了用于蛋白质合成的水凝胶的额外的灵活性，但也可以使目标蛋白质的高密度，均匀的载荷在整个水凝胶，作为目标蛋白质前的水凝胶形成的装载。 内含肽介导的蛋白的水凝胶是高度站ble在水溶液中有小到没有3个月室温后检测的侵蚀。稳定性被保持在一个宽范围的pH（6-10）和温度（4-50℃），并在水凝胶也与有机溶剂相容。这样的水凝胶可用于双球状蛋白质的固定化：绿色荧光蛋白（GFP）和辣根过氧化物酶（HRP）。水凝胶包埋后者蛋白用于在有机溶剂中进行生物催化。

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			Catalog Number
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			Comments
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			Phusion High Fidelity DNA polymerase
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			<td style="width:111px;height:23px;">
			New England BioLabs
			</td>
			<td nowrap="nowrap" style="width:118px;height:23px;">
			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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			<td style="width:175px;height:21px;">
			Luria Bertani
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			<td style="width:111px;height:21px;">
			VWR
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			<td nowrap="nowrap" style="width:118px;height:21px;">
			90003-350
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			<td style="width:175px;height:21px;">
			Bacto Agar Media
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			<td style="width:111px;height:21px;">
			VWR
			</td>
			<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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			<td nowrap="nowrap" style="width:111px;height:21px;">
			VWR
			</td>
			<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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			<td nowrap="nowrap" style="width:118px;height:21px;">
			EM-5720
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			<td nowrap="nowrap" style="width:137px;height:21px;">
			
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			Urea
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			<td nowrap="nowrap" style="width:111px;height:21px;">
			VWR
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			<td nowrap="nowrap" style="width:118px;height:21px;">
			EM-9510
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			<td nowrap="nowrap" style="width:137px;height:21px;">
			
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			Dithiothreitol (DTT)
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			Fisher
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			BP172-5
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			<td style="width:175px;height:21px;">
			Protease Inhibitor cocktail
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			<td nowrap="nowrap" style="width:111px;height:21px;">
			Roche Applied Science
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			11836153001
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			DPBS
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			<td style="width:111px;height:21px;">
			VWR
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			82020-066
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			<td nowrap="nowrap" style="width:137px;height:21px;">
			
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			Brilliant Blue R
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			<td style="width:111px;height:21px;">
			Acros Organics
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			<td style="width:118px;height:21px;">
			A0297990
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			Sodium Azide
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			Fisher
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			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC190380050
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			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
			</td>
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			<td style="width:175px;height:21px;">
			Horseradish peroxidase
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			<td style="width:111px;height:21px;">
			Sigma
			</td>
			<td style="width:118px;height:21px;">
			P8125-5KU
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			<td nowrap="nowrap" style="width:175px;height:21px;">
			N,N-dimethyl-p-phenylene diamine
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			<td style="width:111px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC408460250
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
			</td>
		</tr>
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			<td nowrap="nowrap" style="width:175px;height:21px;">
			phenol
			</td>
			<td style="width:111px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC149340500
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
			</td>
		</tr>
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			<td nowrap="nowrap" style="width:175px;height:21px;">
			tert-butyl hydroperoxide
			</td>
			<td style="width:111px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC180340050
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			n-heptane
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			Acros Organics
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			120340010
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			<td nowrap="nowrap" style="width:137px;height:21px;">
			
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			<td nowrap="nowrap" style="width:137px;height:21px;">[header]</td>
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			<td style="width:175px;height:21px;">
			Shaker/Incubator
			</td>
			<td style="width:111px;height:21px;">
			Fisher Scientific
			</td>
			<td style="width:118px;height:21px;">
			Max Q 6000
			</td>
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synthesis of an intein-mediated artificial protein hydrogel

我们提出了一个高度稳定的蛋白水凝胶由一个分裂蛋白-蛋白催化的转拼反应介导的合成。这种水凝胶的组成部分有两个蛋白嵌段共聚物各自包含有作为拆分内含肽的交联剂，和一个半三聚体蛋白质的一个亚单位。一种高度亲水性的无规卷曲插入嵌段共聚物用于水保持量为1。两个蛋白的嵌段共聚物的混合触发一个内含肽转拼反应，产生具有交联剂的多肽单元在任一端，可以快速自组装成水凝胶。此水凝胶是非常稳定的酸性和碱性条件下，在温度高达50℃，并在有机溶剂中。剪切诱导破裂后的水凝胶迅速改革。成立一个“基座肽”到水凝胶积木使“对接蛋白”标记的靶蛋白的方便结合。所述水凝胶是用组织培养的生长培养基兼容，支持的20kDa的分子的扩散，并且使生物活性的球状蛋白质的固定化。内含肽介导的蛋白水凝胶作为有机溶剂相容的生物催化剂的应用证明了包封的辣根过氧化物酶和确证其活性。

示意图为内含肽介导的蛋白质凝胶的形成是在图1A。水凝胶的基石是蛋白质共聚物CUTA-NpuN（N）和NpuC-S-CUTA（C）（ 图1A，表1）。 NpuN / C是自然分割DnaE的N-/C-fragments内含肽从发菜punctiforme（NPU）。 CUTA是来自激烈horikoshii 4,5稳定的三聚体蛋白质。纯化的N和C中的还原剂DTT存在下的混合导致形成三分之一蛋白-连接产物（j：CUTA-S-CUTA）（ 图1A和1C）...

Watch this Scientific Journal Video about Synthesis of an Intein-mediated Artificial Protein Hydrogel at JoVE.com

Synthesis of an Intein-mediated Artificial Protein Hydrogel

我们提出了一个分裂内含肽介导的蛋白水凝胶的合成。这种水凝胶的组成部分是各含有作为拆分内含肽的交联剂，和一个半三聚体蛋白质的一个亚单位2蛋白共聚物。两个蛋白共聚物的混合触发一个内含肽转拼反应，产生的多肽单元，其自组装成水凝胶。此水凝胶是高度的pH和温度稳定，与有机溶剂相容的，并且很容易地集成功能的球状蛋白。

内含肽介导的人工蛋白水凝胶的合成

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

Research

JoVE Journal

Bioengineering

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

科研

生物工程

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

组合合成和高通量的蛋白质释放的聚合物薄膜和纳米粒子库

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

工程双层凝胶控制升序分化

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

一种简便快速制备磁响应元件的水凝胶基微型机械的添加剂制造技术

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

以自动化和标准化方式制造基于水凝胶的3D培养模型的开源技术平台

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...