Name: pcr mutagenesis, cloning, expression, fast protein purification protocols and crystallization of the wild type and mutant forms of tryptophan synthase
Uploaded: 2020-09-26T15:00:00
Description: Watch this Scientific Journal Video about PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase at JoVE

这项工作得到了美国国家卫生研究院（GM097569）的支持。

1. 快速协议，以净化α和β子和重组α 2 β 2圣TS 综合体
<ol><li>脱氧核糖核酸分包成 pETSUMO 表达载体<ol><li>获得翻译耦合基因（trpA和trpB）编码 α-和 β- 从 细菌沙门氏菌肠 血清石 血清石 克隆在pEBA-10表达载体22的色氨酸合成酶的亚单位。使用 pEBA-10 载体作为 DNA 模板。 注：或者，下面列出的引物可用于放大 来自沙门氏菌肠...

<ol>
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我们成功设计了变异体形式α2β2 β114A、α2β2 0+K167T 和α2β2 S377A + StTS 复合体，用于结构功能相关性研究。最初，我们尝试用以前的纯化协议22净化突变体，这需要α 2 β 2圣TS复合结晶与PEG 8000和精子在净化过程中。虽然结晶率取决于突变形式和复杂物在溶液中的浓度，但很难预测晶体何时以大溶液体积...

色氨酸合成酶（TS）双酶复合物（α 2 β 2）是一种异体酶，催化了细菌、植物和真菌1、2、3中氨基酸L-色氨酸生物合成的最后两步。沙门氏菌肠血清病毒血清素（圣）引起人类和其他动物的严重胃肠道感染。由于人类和高等动物没有 TS（EC 4.2.1.20），S 的抑制。 二甲苯甲酸酯α 2 β 2 TS复合物（α 2 β 2StTS）已被探索为治疗隐孢子虫病和结核病4，生殖器和眼部感染5，以及农业中潜在的除草剂使用的潜在药物目标6。 α - 亚单位通过形成一种氨基苯丙胺陶托默中间体，随后产生 GAP 和内脏3 ， 6的碳碳粘结，促进甘油三磷酸盐（ GAP ）和内脏的碱性。β催化剂位点含有一种通过希夫碱与β-Lys87结合的磷酸杆菌（PLP）共因子分子，在酶β-子单位3、7的反应过程中充当电子汇。β部位通过用惰性剂促进L-Serine侧链羟基的替代，使L-色氨酸和水分子在PLP依赖反应中。圣TS是研究多酶复合物2、3中基质导流和同位素通信的长期模型。TS 的α和β子单元之间的双向同体通信对于同步催化步骤和防止 L-色氨酸合成3期间的内多尔释放是必要的。为了扩大这一努力，我们准备了几个突变体（β-Gln114Ala、β-Lys167Thr和β-Ser377Ala），用于进一步探索圣TS β子单位催化部位酶结构、机制和功能之间的关系。
伊迪丝·迈尔斯研究小组对α 2 β 2圣TS的催化机理进行了详细的研究。早期研究与本地大肠杆菌α 2 β 2TS复合体，重点是净化和定性孤立的α子单元8，9，孤立的β子单元10，11和重组α 2 β 2TS综合体从孤立的子单元12。净化是通过硫酸铵沉淀、样品透析、DEAE-Sephadex色谱、透析和DEAE-Sephadex列12上的第二个色谱圆进行的。在另一个协议中，通过将澄清的细胞裂解物加载到DEAE-Sephadex柱上，然后在Sepharose 4B柱上加载色谱步骤、硫酸铵沉淀和透析13，改进了同一复合物的净化。两种净化协议持续 4-5 天。大肠杆菌α 2 β 2TS复合结晶，但晶体当时不适合X射线衍射。
在一项新的研究中，重组和野生类型的S.型甲基βα甲基重组α 2 β 2圣TS复合体在携带pEBA-10表达载体的大肠杆菌株CB149中过度表达。初步结晶和X射线衍射数据收集和分析的α 2 β 2圣TS综合体报告14。然而，像α 2 β 2圣TS晶体的长而细的针头损害了结构研究。为了收集更好的X射线衍射数据，另一个净化协议被描述为净化野生类型和突变形式的α 2 β 2圣TS复合体15。净化是使用精氨酸和PEG 8，000进入澄清细胞解剖进行初始降水，并通过离心去除一个大的散沉淀物。含有大量α 2 β 2StTS复合物的超高分数在4°C下储存了16-48小时，直到黄色晶体沉淀。晶体被洗净，并广泛透析对不同的缓冲。蛋白质复合物在含有硫酸铵的缓冲器中重新装覆，并透析15。虽然蛋白质结晶取决于溶液中的蛋白质和沉淀物浓度，但很难监测、预测和繁殖溶液中其他突变形式的α2β2StTS 复合物的纯化。此协议具有不使用任何色谱方法的优势：然而，缺点是结晶、透析和重新装覆所需的较长的净化时间，通常需要 5-7 天。为了获得适合X射线数据采集的晶体，使用蛋白质浓度、温度、沉淀剂（PEG 4，000、6，000和8，000）和添加剂（CaCl 2、MnCl2、ZnCl2、尸体、普氏精氨酸、精子或精氨酸）的组合和变异对600多个结晶条件进行了评估。晶体具有更好的晶体形式，在含有 12% PEG 8，000 和 2mM 精子的条件下生长得更快。结晶在 25 °C 时比在 4、30 或 42 °C 更有利，并在15天内增长到最大尺寸。据报道，当时（1996-1999年）16、17、18、19、20、21和许多其他结构已公布α 2 β 2个圣TS晶体结构。
在这里，主要目的是提出替代协议，以净化色氨酸合成酶和优化蛋白质结晶。目前的工作表明，在净化野生类型孤立的α子单元（α圣TS），孤立的β子单位（β圣TS），重建α 2 β 2圣TS综合体从孤立的亚单位，野生类型和突变形式的α 2 β 2圣TS综合体。 由于净化时间显著缩短，结晶和低温保护得到优化，与以往协议相比，其优势相当大。在这项工作中设计的α 2 β 2StTS复合体的突变形式已经结晶，接近野生类型形式所用的条件。然而，为了获得质量足够的大单晶，在接近原子分辨率的结构确定方面，必须进行精细的结晶优化。迄今为止，蛋白质数据库（PDB）中存存的色氨酸合成酶晶体结构有134个，分别占细菌、古生物和真核生物晶体结构的101个、31个和2个晶体结构。不错的是，73个结构属于S.肠血清石，5个晶体结构的α 2 β 2圣TS综合体有分辨率限制高于1.50安格斯特罗姆。毫不奇怪，我们的研究小组准备了 4 分 5 秒（PDB ID：5CGQ 在 1.18 + ， 4HT3 在 1.30 +， 4HPJ 在 1.45 +， 6DZ4 在 1.45 + 分辨率）。α 2 β 2圣TS 复合体的突变形式的精炼晶体结构预计将为了解 L-色氨酸合成中所涉及的基本氨基酸残留物所起的机制和作用提供新的见解。

<table>
	<tbody>
		<tr>
			<td>15 mL 10 kDa filter</td>
			<td>MilliporeSigma</td>
			<td>UFC901024</td>
			<td>centrifugal filter unit</td>
		</tr>
		<tr>
			<td>15 mL 100 kDa filter</td>
			<td>MilliporeSigma</td>
			<td>UFC910024</td>
			<td>centrifugal filter unit</td>
		</tr>
		<tr>
			<td>2 mL cryogenic vials</td>
			<td>Corning</td>
			<td>CLS430489</td>
			<td>Cryogenic vials</td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-141</td>
			<td>microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>24-well Cryschem Plate</td>
			<td>Hampton Research</td>
			<td>HR3-158</td>
			<td>24-well sitting drop plates</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Fisher Scientific</td>
			<td>O3446I-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>50 mL centrifuge conical tubes</td>
			<td>Thermo Scientific</td>
			<td>12-565-270</td>
			<td>centrifuge conical tubes</td>
		</tr>
		<tr>
			<td>AB15 ACCUMET Basic</td>
			<td>Fisher Scientific</td>
			<td>13-636-AB15</td>
			<td>pH meter</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP1356-100</td>
			<td>Agarose gel</td>
		</tr>
		<tr>
			<td>ammonium sulfate</td>
			<td>Fisher Scientific</td>
			<td>A702-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760-5</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Bacterial incubator</td>
			<td>Fisher Scientific</td>
			<td>S35836</td>
			<td>incubator.</td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>New England Biolabs</td>
			<td>R0136S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>bicine</td>
			<td>Fisher Scientific</td>
			<td>BP2646100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Branson 450 Digital Sonifier</td>
			<td>Brason</td>
			<td>B450</td>
			<td>Cell disruptor</td>
		</tr>
		<tr>
			<td>Cesium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP210-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Cesium hydroxide</td>
			<td>Acros Organics</td>
			<td>AC213601000</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Fisher Scientific</td>
			<td>BP904-100</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>D1391</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F530S</td>
			<td>HF polymerase</td>
		</tr>
		<tr>
			<td>dNTP Set</td>
			<td>Invitrogen</td>
			<td>10-297-018</td>
			<td>dNTPs set</td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>New England Biolabs</td>
			<td>R0101S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Fisher Scientific</td>
			<td>S311-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td height="42">Excella E25R Orbital Shaker</td>
			<td>Eppendorf New Brunswick</td>
			<td>M1353-0004</td>
			<td>Orbital incubator</td>
		</tr>
		<tr>
			<td>GE AKTA Prime Plus</td>
			<td>GE Healthcare</td>
			<td>8149-30-0004</td>
			<td>FPLC</td>
		</tr>
		<tr>
			<td>Gel Extraction Kit</td>
			<td>Invitrogen</td>
			<td>K210012</td>
			<td>DNA purification kit</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>HindIII</td>
			<td>New England Biolabs</td>
			<td>R0104S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>His-Trap columns</td>
			<td>GE Healthcare</td>
			<td>GE17-5255-01</td>
			<td>5 mL Histrap column</td>
		</tr>
		<tr>
			<td>imidazole</td>
			<td>Fisher Scientific</td>
			<td>O3196-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0392</td>
			<td>Inducer</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Kelvinator Series-100</td>
			<td>Kelvinator</td>
			<td>discontinued</td>
			<td>Ultra low freezer</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td>Liquid broth</td>
		</tr>
		<tr>
			<td>Luria Bertani agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425-2</td>
			<td>Solid broth</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>NcoI</td>
			<td>New England Biolabs</td>
			<td>R0193S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Ni-NTA affinity beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>R90115</td>
			<td>Ni-NTA agarose beads</td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>Fisher Scientific</td>
			<td>BP233-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Fisher Scientific</td>
			<td>44-865-0</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>pyridoxal phosphate</td>
			<td>Acros Organics</td>
			<td>AC228170010</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>S-200 HR</td>
			<td>Cytiva</td>
			<td>45-000-196</td>
			<td>Size exclusion column</td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sorvall RC-5B centrifuge</td>
			<td>Sorvall</td>
			<td>8327-30-1004</td>
			<td>Floor cetrifuge</td>
		</tr>
		<tr>
			<td>Spermine</td>
			<td>Acros Organics</td>
			<td>AC132750010</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Superdex 200 prep grade</td>
			<td>Cytiva</td>
			<td>45-002-491</td>
			<td>Size exclusion column</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td>DNA liagse</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Ubl-specific protease 1</td>
			<td>Thermo Scientific</td>
			<td>12588018</td>
			<td>SUMO Protease</td>
		</tr>
	</tbody>
</table>

pcr mutagenesis, cloning, expression, fast protein purification protocols and crystallization of the wild type and mutant forms of tryptophan synthase

对来自沙门氏菌的色氨酸合成酶（TS）双酶复合物（α 2 β 2 TS）进行了结构研究，以更好地了解其催化机制、同位素行为以及PPLP依赖酶中基质向产品酶转化的细节。在这项工作中，一种新的表达系统，以产生孤立的α和孤立的β子单位允许净化大量的纯子单位和α 2 β 2圣TS综合体从孤立的亚单位在2天内。净化由亲和度色谱进行，然后是亲和力标记的、硫酸铵沉淀和大小排除色谱（ SEC ）。为了更好地了解酶β部位的关键残留物的作用，在以前的结构研究中进行了现场直接突变。另一个协议被创建，以净化野生类型和突变体α 2 β 2圣TS综合体。使用硫酸铵分馏和 SEC 的简单、快速和高效的协议允许在一天内净化α2β2StTS 复合物。与之前使用 PEG 8000 和精氨酸沿净化协议对同一复合物进行净化的先前协议相比，本文中描述的两种净化协议都具有相当大的优势，使α2β2StTS 复合体结晶。野生类型和某些突变形式的结晶发生在略有不同的条件下，损害了使用PEG 8000和精子的一些突变体的净化。为准备适合X射线晶体学研究的晶体，我们努力优化结晶、晶体质量和低温保护。这里提出的方法通常应适用于净化色氨酸合成酶亚单位和野生类型和突变α2β2圣TS复合物。

色氨酸合成酶的α和β子单位的净化 沙门氏菌斑马素合成酶的α子联盟（+StTS） 和β子联盟 （βStTS） 在修改后的 pET SUMO 载体中进行了分克隆。图1A显示了两个强频段的代表性SDS-PAGE结果，对应于他的6-SUMO-α圣TS（车道+ON）和他的6-SUMO-β圣TS（车道+ON）融合蛋?...

Watch this Scientific Journal Video about PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase at JoVE

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

本文介绍了一系列连续的方法来表达和净化 沙门氏菌色氨酸 合成酶，使该协议成为一天中净化蛋白质复合物的快速系统。覆盖的方法包括现场定向突变、 大肠杆菌中的蛋白质表达、亲和色谱、凝胶过滤色谱和结晶。

PCR 突变体、克隆、表达、快速蛋白质纯化协议和色氨酸合成酶野生类型和突变形式的结晶

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Expression, Purification, and Antimicrobial Activity of S100A12

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

重组蛋白表达，结晶和生物物理研究<em&gt;芽孢杆菌</em&gt; - 保守的核苷酸焦磷酸化酶，BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

分子生物学实验室中大分子结晶的自动化协议

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

Expression and Purification of Mammalian Bestrophin Ion Channels

哺乳动物 Bestrophin 离子通道的表达与纯化

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

真核硼酸背液转运体的表达、增溶及纯化

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography (Video) | JoVE 

高通量筛选以获得用于蛋白质晶体学的晶体命中

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...