这项工作得到了美国国家卫生研究院（GM097569）的支持。

<ol>
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作者没有什么可披露的，也没有宣布没有相互竞争的经济利益。

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出现。结晶可以在长时间（48-96小时）后实现，或在结晶启动15后增加额外的PEG 8000。
不幸的是，本作品中提出的α 2 β 2StTS复合体的突变形式没有使用此协议成功净化，因为它们未能在协议的初始步骤中结晶，损害了结晶研究。因此，我们创建了一个简单高效的净化方案，包括硫酸铵分馏和大小排除色谱，使野生类型和突变形式的高产量α 2 β 2圣TS 复合体。此协议比之前的协议（5-7 天） 15、22 更快（1-2 天），并且可重复，因为没有结晶要求和协议故障排除沿净化。此外，我们还创建了新的表达结构和协议，以净化大量α子组、β子组和重组α2β2StTS 综合体与隔离体。
未来的应用包括重组野生类型和突变子单元，以进行功能和结构研究。突变α2β2 β114A，α2β2 βK167T，α2β2 βS377A 复合结晶的条件包含更高的精子浓度 （4-8 mM） 相比，野生类型形式 （2 mM）.。因此，通过改变沉淀物和缓冲pH值的浓度来提高蛋白质晶体的质量是值得的。单个大晶体形式随机生长在二头肌缓冲溶液 （pH 7.6-8.0） 包含 6-11% PEG 8，000。本研究中描述的方法将用于制备野生类型和变异形式的晶体结构，α 2 β 2复合体在α和β活跃部位具有不同的配体，模仿不同的中间体和过渡状态，参与将内单体和血清转换为色氨酸。这些突变体的晶体结构预计将为L-色氨酸合成中的关键残留物所起的机制和作用提供新的见解。

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>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）融合蛋白。本著作中描述的净化协议允许在 2 天内分别对两个子单位进行净化。第一天用于通过 Ni - NTA 亲和色谱、硫酸铵沉淀以及他的 SUO 标签、去除他的 SUO 标签痕迹和蛋白质浓度来净化每种蛋白质。图1B和1C分别显示了α子组和β子组净化的代表性SDS-PAGE结果。第二天，集中α子单元、β子单元和α和β子单位的α 2 β 2 StTS复合体被加载在大小排除色谱柱上。图 1D显示了S-200 HR大小排除列中 1ST TS、βStTS 和α2β2StTS 复合体的典型脱口而出了特征。图 1E显示了收集的峰值分数的代表性 SDS-PAGE 结果。最纯净的峰值分数被集中，浓缩，α 2 β 2圣TS复合物用于蛋白质结晶研究。
纯化野生类型和突变体α2β2圣TS 复合体 本文描述了另一种快速高效的程序，以净化α 2 β 2S.硫化钛色氨酸合成酶复合物的野生类型和突变形式。图2显示了pEBA-10结构的表示，其中包含用于α的野生类型转化耦合基因（trpA和trpB）编码，以及β-子单位22。图3中描绘了产生α 2 β 2圣TS 复合体突变形式的两步 PCR 突变方案。
pEBA10中突变α 2 β 2圣TS复合体的编码区域通过DNA测序得到确认，用于转化大肠杆菌菌株CB149细胞26。α 2 β 2圣TS 复合体的野生类型和突变形式过度表达，重组蛋白在 1-2 天内成功纯化。硫酸铵在室温下分馏很容易从异质表达系统中去除大部分污染物蛋白（图4A，车道20P，30P和40S）。在图 4B中显示了具有相对椭α2β2StTS （143.06 kDa） 复合物的具有相对椭形位置的代表性椭形配置文件。排除峰值分数的纯度在汇集前进行了 SDS-PAGE 分析（图 4C）。
优化野生类型和突变α2β2色氨酸合成酶复合结晶 野生类型和突变α2β2StTScomplex 在 15 毫克毫升-1的阿里奎特被用来设置 24 井坐滴板。通常，由 5 μL 蛋白质溶液和等量的储层溶液组成的液滴与 500 μL 的储液溶液（图 5）均等。精子需要结晶的野生类型和突变α 2 β 2圣TS复合物22。虽然精子结晶野生类型的最终浓度为2mM，但在此工作中，精子结晶突变复合物的浓度略高（4-8mM）。
大单晶是通过精细的结晶优化获得的，变化PEG 8000（6-11%）和二甲酸缓冲pH值。不同形态的晶体在2-5天内出现，晶体在两周内长到全尺寸（图6）。在X射线衍射数据收集之前，晶体被浸泡在低温保护液中（储液缓冲器含有高达30%的二甲基硫氧化物）。优化的过程产生了质量晶体，适合以接近原子分辨率的X射线衍射测量。
X 射线衍射数据分析 野生型α 2 β 2StTS复合物的晶体结构是用本文中描述的方法制备的，X射线衍射数据以接近原子分辨率的速度收集。晶体浸泡在含有F9抑制剂（2-（[4-）（三氟甲基）苯基+硫化物+氨基）乙基二氢磷酸盐）和L-色氨酸的低温保护溶液中。
通过以0.5°的增量旋转晶体360°，在高级光源（伯克利-CA）的SIBYLS同步光束线12.3.1上收集了完整的X射线衍射数据集。处理了X射线衍射强度，并将数据收集统计数据汇总在表1中。对称分析表明，该晶体属于单气候空间组C2。单位单元参数为= 182.55，b = 59.30，c = 67.37+，α = 90.00，β = 94.82，γ = 90.00°。 马修斯系数的计算值（Vm = 2.57 =3 Da-1）表明晶体的不对称单元中存在一个 TS 异质分子 （+St TS），溶剂含量为 52.08%35，36。 所有 X 射线数据都是在低温 （100 K） 下收集的，以提高衍射质量并减少辐射衰变。复杂中α2β2StTS 晶体结构是使用野生类型 + StTS 模型在复合物中与α部位的抑制剂 F9 和金属协调部位的钚离子 （PDB ID 代码: 4HT3） 解决的。最终坐标文件和结构因素存入PDB与加入代码5CGQ（图7A）。野生型的晶体结构α 2 β 2圣TS复合物与抑制剂F9在酶α部位（图7B），在金属协调点（图7C），共因子皮里多克斯 5'-磷酸盐共价粘结到βLys87（图7D），酶β位（图7E）的产品L-色氨酸以1.18安斯特伦分辨率解决。型号 5CGQ 是迄今为止存放在 PDB 中的最高分辨率α2β2StTS 晶体结构。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61839/61839fig1v2.jpg"> 图1:净化α和β单位和α 2 β 2圣TS综合体。（A） 重组蛋白表达。12% SDS-PAGE 凝胶的隔夜表达配置文件的相扑α圣TS （+ON） 和相扑β圣TS （+ON） 后，IPTG 感应在 30 °C （α/0 之前 IPTG 诱导）。（B， C）Ni-NTA 亲和色谱，然后是硫酸铵沉淀（60% 饱和度），（S） 澄清粗提取物 （FT1） Ni-NTA 列通过样品 （W） 柱洗样品 （E） eluate 样品 （60S） 和 （60P） 超高沉淀分数后高速离心， 分别 （D） SUMO 蛋白酶消化产品 （FT2） Ni-NTA 列通过包含无标签的α圣TS 或β圣TS 子联盟的样品。（D） α圣TS 子联盟 （28.67 kDa）、 β圣TS 子联盟 （42.86 kDa） 和 α2的逃学概况 β2圣TS 复合物 （143.06 kDa） 与 HiPrep 16/60 Sephacryl S-200 HR 大小排除色谱柱。每个运行是单独执行的。（E） 每个单色谱中排除峰值分数的 SDS-PAGE 凝胶。15% 的SDS-PAGE 凝胶准备分析α2β2StTS 综合体和α圣TS 子组，12% SDS-PAGE 凝胶准备分析圣TS子组的β。巷 MW，kDa 中的分子量标记。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61839/61839fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61839/61839fig2v2.jpg"> 图2:构建pEBA-10的表示。（A） 表示从沙门氏菌肠血清素血清素（杨，艾哈迈德等人）中编码α的野生类型转化耦合基因（trpA和trpB）和β-亚单位。（B） 载体包含安非他林耐药性（amp）基因、复制源（ori）、在没有IPTG诱导剂的情况下更好地关闭乳酸促进剂的lacIq基因，以及LacI抑制促进剂。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61839/61839fig02large.jpg" target="_blank"></a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61839/61839fig3v2.jpg"> 图3:两步PCR突变协议的总体表示。pEBA-10载体被用作DNA模板。第一轮PCR是用引物 TS-FW-NcoI 和 MUT-REV（包含突变的反向引物）准备的，以生成第一个片段和引物 TS-Rev-SacI 和 MUT-FW（包含突变的前进引物）生成第二个片段。碎片被凝胶纯化和等价结合，热变性和退火。重组链用聚合酶和脱氧核糖核酸延长。第二轮PCR是用引物 TS-FW-NcoI 和 TS-Rev-Saci 准备的。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61839/61839fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61839/61839fig4v2.jpg"> 图4:α 2 β 2StTS复合体的野生类型和突变形态的净化。（A） 使用20、30、40和50%的硫酸铵饱和在室温下收集的样品的12%SDS-PAGE凝胶:（CE）粗提取物（S）和（P）高速离心后超高沉淀分馏。（B） α 2 β 2圣TS （143.06 kDa） 复合物的椭形配置文件，带有 HiPrep 16/60 Sephacryl S-200 HR 尺寸排除色谱柱。（C） 12% SDS-PAGE 凝胶图片的排除峰值分数。巷 MW， kDa 中的分子量标记 （精度加蛋白质未染色标准， 生物拉德） 。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61839/61839fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61839/61839fig5v2.jpg"> 图5:野生类型和变异形态的结晶优化α 2 β 2圣TS复合体。晶体生长在50mM比辛-CsOH缓冲器中，含有50mM CsCl2。聚乙烯乙二醇8000（6-11%）和精氨酸（2-8mM）的浓度各不相同，以获得单个大晶体形式，通过X射线蛋白晶体学进行结构研究。（A） 比辛-C索，pH 7.6。（B） 比辛-C索，pH 7.8。（C） 比辛-C索，pH 8.0。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61839/61839fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61839/61839fig6v2.jpg"> 图6:野生晶体光谱仪和α 2 β 2圣TS复合体的突变形态。晶体的形态不同，但它们属于空间C2组。两周后，晶体在最终条件下长到全部尺寸。晶体大小约 0.20 x 0.15 x 0.10 mm。（A-D）PLP 全息晶体在野生类型形式（列 A）的金属协调部位与氦离子复合，突变形式α2β2 β114A （列 B），α2β2 β167T （列 C）， 和α2β2 βS377A （列 D）。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61839/61839fig06large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61839/61839fig7v2.jpg"> 图7:晶体结构的整体可视化和晶体结构优化后获得的电子密度图的验证。（A） 野生型α 2 β 2圣TS复合物的晶体结构，酶α部位（黄色）、金属协调部位（蓝色）、共生器Pyrid的抑制剂F9草5'磷酸盐共价粘结到+Lys87（绿色），产品L-色氨酸在酶β位（青色）在1.18安斯特伦分辨率。α子组为浅蓝色，β子组为鲑鱼色。（B-E）电子密度图轮廓在1.0 r.m s水平周围（B） 抑制剂F9（C） 二恶英离子（D） 磷酸二恶英 - 5+磷酸盐 ， 和（E） L - 色氨酸。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61839/61839fig07large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">数据收集和处理</td>
		</tr>
<tr>
<td>X 射线源 / 光束线</td>
			<td>ALS 光束线 12.3.1</td>
		</tr>
<tr>
<td>波长 （+）</td>
			<td>10,000</td>
		</tr>
<tr>
<td>分辨率 （+）</td>
			<td>40.00 - 1.18 (1.24 - 1.18)</td>
		</tr>
<tr>
<td>反射总数</td>
			<td>2151280 (252941)</td>
		</tr>
<tr>
<td>总数唯一反射</td>
			<td>231646 (32187)</td>
		</tr>
<tr>
<td>用于索引、缩放和合并的空间组</td>
			<td>C 1 2 1</td>
		</tr>
<tr>
<td colspan="2">单元格尺寸</td>
		</tr>
<tr>
<td>a， b， c （+）</td>
			<td>182.55, 59.30, 67.37</td>
		</tr>
<tr>
<td>α, β, γ (°)</td>
			<td>90.00, 94.82, 90.00</td>
		</tr>
<tr>
<td>马赛克</td>
			<td>0.61</td>
		</tr>
<tr>
<td>马修斯卷 VM（+3 Da-1）</td>
			<td>2.57</td>
		</tr>
<tr>
<td>雷米亚斯 （%）</td>
			<td>8.6 (93.0)</td>
		</tr>
<tr>
<td><I/σ（I）></td>
			<td>14.7 (3.0)</td>
		</tr>
<tr>
<td>CC1+2 （%）</td>
			<td>0.999 (0.778)</td>
		</tr>
<tr>
<td>完整性 （%）</td>
			<td>98.6 (94.2)</td>
		</tr>
<tr>
<td>多样性</td>
			<td>9.3 (7.9)</td>
		</tr>
<tr>
<td colspan="2">精细化统计</td>
		</tr>
<tr>
<td>工作/自由 （%）</td>
			<td>14.04 / 16.05</td>
		</tr>
<tr>
<td>RMSD 债券长度 （+）</td>
			<td>0.0120</td>
		</tr>
<tr>
<td>RMSD 键角 （+）</td>
			<td>14,059</td>
		</tr>
<tr>
<td>拉马坎德兰青睐</td>
			<td>515 (96.44%)</td>
		</tr>
<tr>
<td>拉马坎德兰允许</td>
			<td>16 (3.00%)</td>
		</tr>
<tr>
<td>拉马尚德兰不允许</td>
			<td>3 (0.56%)</td>
		</tr>
</tbody></table>表1:数据收集和处理。括号中的值用于外壳。

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 ...

pcr-mutagenesis-cloning-expression-fast-protein-purification

Nanyang Technological University

Research

JoVE Journal

Biochemistry

4.3K 次观看.  University of California-Riverside. 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。

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

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

科研

生物化学

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

X射线晶体学研究热木体马里蒂玛M42氨基肽TmPep1050的寡聚态过渡

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

生物工程

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

生物学

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

免疫与感染

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

使用无细胞系统对用于蛋白质原型的最小线性模板进行扩增的快速酶促方法

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

链球菌中高分子质量蛋白的纯化

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.