Name: purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli
Uploaded: 2015-12-19T14:00:00
Description: Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

The authors would like to acknowledge Natalie Doude and David Westerway (University of Alberta) for providing us Sho cDNA, Christophe Chevalier (INRA) for plasmid construction, Michel Br&#233;mont (INRA) for providing us BL21Sol bacteria; Christine Longin (INRA) for TEM assistance and Edith Pajot-Augy (INRA) for comments and proofreading.

1.代质粒和Shadoo表达注：基因编码鼠Shadoo蛋白（Shadoo 25-122），亚克隆到表达载体pET-28 11。该克隆转化 E. 大肠杆菌 SoluBL21菌株，表达Shadoo蛋白质与N-末端六组氨酸标签，（HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG）如先前11说明。质粒可以从作者索取。 Shadoo的​​分子量从它的氨基酸序列计...

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一个简单的，高效的协议为表达和大量重组小鼠Shadoo蛋白的纯化给出。所描述的方法可以成功溶解和（他的6）-tagged重组Shadoo蛋白的纯化。重要的是，如在标题所指出的，当在原核细菌大肠杆菌表达大肠杆菌 Shadoo积聚在包涵体。之前Shadoo纯化，细菌必须通过超声处理在含有的Triton X-100的缓冲液裂解。接着包涵体可溶于8M尿素变性溶液。此外，整个纯化过程必须在8M尿素缓冲液?...

朊病毒神经变性疾病，其中包括牛海绵状脑病在牛，羊瘙痒在绵羊和Creutzfeldt-Jakob病在人类，是致命的，无法治愈的。朊病毒病的特征在于细胞朊病毒蛋白的构象变化主要由α螺旋的，进入交叉β-片富含淀粉样蛋白构象1,2-。交叉β-结构包含密集和高度有序的结晶状β片具有稳定的氢键3,4。朊病毒淀粉样蛋白可以自我复制由于β链的生长边缘提供招募的模板和转换单体蛋白质单元。 据“唯一蛋白质”假说，朊病毒蛋白的淀粉样蛋白构象是唯一的感染因子。然而，一些其它生物分子已经被提出是必不可少的朊病毒病。例如，细胞膜被认为是一个地方，conversi上发生与一些负电荷的脂质已经显示出增强了转换过程5,6。此外，某些蛋白质也可以是参与朊病毒病症。具体来说，有朊蛋白家族的另外两名成员：Doppel和Shadoo 7,8。 Doppel具有相对刚性的结构稳定化与二硫醚桥，未能自聚合和聚合到淀粉样蛋白原纤维9。与此相反，类似于朊病毒蛋白，Shadoo可以采用一个β片层富集结构和自缔成淀粉样蛋白纤维的结构。它表明，Shadoo可在天然条件下或在结合带负电荷的膜10,11原纤化。转换Shadoo成淀粉样纤维可以与病理相关联。事实上，通过淀粉样蛋白样结构引起疾病的机理知之甚少。 Shadoo熊疏水结构域（HD）等一系列串联精氨酸/甘氨酸的重复类似的牛逼Ø朊蛋白（ 图1A）的N末端 ​​部分。如朊病毒蛋白的N-端部分，Shadoo高度正电，似乎是一个本地非结构化蛋白11,12。 Shadoo似乎是功能上相关的朊病毒疾病，因为它可以直接绑定朊病毒蛋白。另外，它的表达被向下期间朊病毒病理13,14调节。然而，Shadoo的​​朊病毒疾病中的作用尚未建立。 我们开发了一种质粒携带鼠标Shadoo基因的编码序列。将质粒用于转化大肠杆菌大肠杆菌生产N端他的6融合Shadoo蛋白质。该表达系统是公认的在我们的实验室和我们正在进行的项目6,15,16常用。为Shadoo的表达的一个重要问题是大肠杆菌的选择大肠杆菌菌株 。而感受态BL21细菌菌株通常用于重组亲表达teins Shadoo已成功表达并只从转化SoluBL21菌株纯化。 SoluBL21主管E.大肠杆菌是BL21宿主菌株的改进的突变体，用于生产蛋白质的表达在父BL21给没有可检测的可溶性产物开发的。 Shadoo的SoluBL21 大肠杆菌高效表达大肠杆菌导致的蛋白质的积累中包涵体。作为一般特征，当非天然蛋白质高表达在大肠杆菌大肠杆菌 ，这种蛋白质往往积聚在不溶性包涵体。 Shadoo大概粒料通过非共价的疏水性或离子相互作用（或两者的组合），以通过折叠到不同程度的蛋白质形成的高度富集的动态结构。由此，纯化包括至少两个步骤：（i）从在变性介质其它生物分子的蛋白的选择性分离，以及（ii） 使用体外折叠纯化蛋白的复性技术。 Shadoo的​​选择性分离物在含有8M尿素（或者6M胍-HCl）的缓冲溶液来实现。 （ⅰ）复性在酸性pH溶液中，得到的非结构化单体Shadoo蛋白质，或（ii）复性pH值≥的溶液7，得到Shadoo聚合：尿素去除和蛋白质的复性可以通过应用不同的协议来实现成稳定的淀粉样蛋白纤维特性交叉β折叠基序。

<table>
	<tbody>
		<tr>
			<td>AKTA FPLC&#160;</td>
			<td>GE, Amersham</td>
			<td># 1363</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/60 Superdex</td>
			<td>GE Healthcare</td>
			<td>17-1069-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap IMAC</td>
			<td>GE Healthcare</td>
			<td>GE17-0920-05</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep26/10 Desalting column</td>
			<td>GE Healthcare</td>
			<td>GE17-5087-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SoluBL21 Competent E. coli</td>
			<td>Genlantis</td>
			<td>C700200</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-28b+</td>
			<td>Novogen</td>
			<td>69865-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Broth medium</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Biophotometar</td>
			<td>Eppendorf</td>
			<td>550507804</td>
			<td></td>
		</tr>
		<tr>
			<td>Trito X-100</td>
			<td>Life Technologies</td>
			<td>85111</td>
			<td></td>
		</tr>
		<tr>
			<td>NiSO4</td>
			<td>Sigma-Aldrich</td>
			<td>656895</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Gdn-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr>
			<td>protein size marker&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>26620</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli

大肠杆菌表达系统是一种强大的工具，用于生产重组真核蛋白。我们用它来生产Shadoo，属于家庭朊病毒蛋白。作为包涵体被描述为（他）6 -tagged重组Shadoo纯化的色谱法表示。包涵体溶解于8M尿素和结合到镍离子-charged柱进行离子亲和层析。结合的蛋白质通过咪唑的梯度洗脱。含有Shadoo蛋白质级分进行尺寸排阻色谱法，以获得高度纯化的蛋白质。在最后的步骤纯化Shadoo脱盐以除去盐，脲和咪唑。重组Shadoo蛋白是用于在朊病毒病发生的蛋白质构象疾病的生物物理和生物化学研究的重要试剂。许多报告表明，朊病毒神经退行性疾病，从刺的沉积起源乐，有序的淀粉样纤维。描述如何纤化Shadoo成淀粉样纤维在酸性和中性样品协议/碱性pH值呈现。关于如何产生和原纤化Shadoo可以促进研究在实验室中工作的朊病毒疾病，因为它允许生产的快速和低成本的方式大量蛋白质的方法。

每升细菌培养物中获得约5-10毫克纯化Shadoo蛋白质。两步净化需要获得高纯度的重组Shadoo。第一步骤是通过亲和层析利用Ni 2+ -charged柱，其保存His-标记的蛋白进行的。洗脱级分进行SDS-PAGE和用考马斯亮蓝（图5A）。含有Shadoo蛋白的级分汇集并通过大小排阻层析其中根据其大小分离蛋白质（Shadoo分子量为约12 kDa的）进一步纯化。在洗脱收集的级分通过SDS-PAGE /考马斯分析（</st...

Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

纯化及复性的淀粉样纤维（他）<sub&gt; 6</sub&gt; -tagged重组Shadoo蛋白表达的包涵体<em&gt;大肠杆菌</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

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Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

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Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...