Name: pcr mutagenesis, cloning, expression, fast protein purification protocols and crystallization of the wild type and mutant forms of tryptophan synthase
Uploaded: 2020-09-26T15:00:00.000+00:00
Duration: 9 min 31 s
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。

<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...

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?...

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

Research

JoVE Journal

Biochemistry

4.2K 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

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

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

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

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

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

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 ...

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 ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

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 ...

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that has been crystallized in various conformations. Detailed functional information may nonetheless be obtained from isolated recombinant domains. The engineered (Trp552Leu and Tyr587Trp) recombinant nucleotide-binding domain (N-domain) displays fluorescence quenching upon ligand binding. An extrinsic fluorophore, namely, 8-anilino-1-naphthalene sulfonate (ANS), binds to the nucleotide-binding site via electrostatic and hydrophobic interactions with Arg, His, Ala, Leu, and Phe residues. ANS binding is evidenced by the increase in fluorescence intensity when excited at a wavelength (&#955;) of 370 nm. However, when excited at &#955; of 295 nm, the increase in fluorescence intensity seems to be coupled to the quenching of the N-domain intrinsic fluorescence. Fluorescence spectra display a F&#246;ster resonance energy transfer (FRET)-like pattern, thereby suggesting the presence of a Trp-ANS FRET pair, which appears to be supported by the short distance (~20 &#197;) between Tyr587Trp and ANS. This study describes an analysis of the Trp-ANS FRET pair by Trp chemical modification (and fluorescence quenching) that is mediated by N-bromosuccinimide (NBS). In the chemically modified N-domain, ANS fluorescence increased when excited at a &#955; of 295 nm, similar to when excited at a &#955; of 370 nm. Hence, the NBS-mediated chemical modification of the Trp residue can be used to probe the absence of FRET between Trp and ANS. In the absence of Trp fluorescence, one should not observe an increase in ANS fluorescence. The chemical modification of Trp residues in proteins by NBS may be useful for examining FRET between Trp residues that are close to the bound ANS. This assay will likely also be useful when using other fluorophores.

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

ANS binds to the Ca2+-ATPase recombinant N-domain. Fluorescence spectra display a FRET-like pattern upon excitation at a wavelength of 295 nm. NBS-mediated chemical modification of Trp quenches the fluorescence of the N-domain, which leads to the absence of energy transfer (FRET) between the Trp residue and ANS.

重组卡2 +- 研究色氨酸 - ANS FRET 的色氨酸残留物的化学修饰 - ATPase N 域

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that has been crystallized in various conformations. Detailed functional ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

生物学

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

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 ...

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

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热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到