Name: thermostabilization, expression, purification, and crystallization of the human serotonin transporter bound to s-citalopram
Uploaded: 2016-11-27T15:00:00.000+00:00
Duration: 12 min 21 s
Description: Watch this Scientific Journal Video about Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram at JoVE.com

We thank D. Cawley for generating monoclonal antibodies. We thank A. Penmatsa and K. Wang for sharing ideas and expertise developed from the dopamine transporter. L. Vaskalis for assistance with figures, H. Owen for help with manuscript preparation and other Gouaux laboratory members for helpful discussions. J.A.C. has support from a Banting postdoctoral fellowship from the Canadian Institutes of Health Research. E.M.G. is supported by the National Science Foundation Graduate Research Fellowship. We are particularly grateful to Bernie and Jennifer LaCroute for their generous support, as well as for funding from the NIH (5R37MH070039). E.G. is an investigator of the Howard Hughes Medical Institute.

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The authors have nothing to disclose.

由生物物理技术膜蛋白结构的测定仍然是许多医学显著转运，受体和通道11是一项艰巨的任务。我们在下面分享了约束向S -citalopram人血清素转运蛋白的结构测定制定了详细的专业知识。我们预计，这些方法将是决定在其他构象状态以及其他困难的膜蛋白的结构SERT的结构非常有用。此外，这里所描述的生物化学技术也可以用来研究在洗涤剂纯化SERT和近天然脂质环境的功能。 SERT结晶铰接后的多种工具和技术的发展。首先，在转运热稳定性的改善产生SERT变体是乖巧的继起膜6转运提取各种洗涤剂胶束。二，使用在整个纯化和结晶的高亲和力配体小号 -citalopram进一步改善的稳定性和降低的构象的异质性。第三，允许生产的2周短时间内大量SERT的BacMam表达系统7，促进两个免疫和结晶的发展。最后，发展战略，以选择识别允许的8B6抗体的发现促进SERT抗体复合物秩序井然包装成晶体的3D表位的高亲和力的抗体。 有许多的这往往在整个协议发生的关键步骤和试剂，以及常见的问题。第一，高滴度SERT P2病毒的产生可能是有问题。加入低浓度的P 1病毒的如在本协议中所述生成的P2病毒通常减轻此问题，并在那里在P2病毒滴度是低的情况下，P3病毒可制成全光照G病毒在0.0001的MOI。用滴度小于1×10 8个病毒颗粒的病毒/ mL的不应使用，并且几乎总是导致低蛋白产量。对于表达，HEK293S GnTI -细胞被选择，因为它们缺乏Ñ-acetylglucosaminyltransferase我活性，因此不能合成复合N- -glycans，代替只生产高甘露糖Ñ-glycans。 EndoH的裂解ñ -连接高甘露聚糖的糖基化在胞外环2（EL2）两个网站，留下附着于天冬酰胺的N -acetylglucosamine。 氮消化-连接的糖减少EL2的表面熵这对结晶有可能很重要。对于抗体的产生，所述SERT 集成电路应用于免疫。 GFP是高度免疫原性的12和不应该被用作融合标记来产生抗体，因为它是难以完全通过SEC除去。 SERT的柔性N-和C-末端也没有包含在构建体中，以避免对这些区的抗体。小鼠可以重新构成蛋白质的30微克进行免疫;继续免疫小鼠直到抗体的高血清浓度可以被检测和产生杂交瘤细胞如所述13。一个thermostabilized结构通常是用于免疫接种的最佳选择;如果转运性能良好，并且保留在纯化生物活性，这往往是足以提高抗体。该8B6抗体产生抗WT SERT。结晶，从SEC含有单分散复杂的由FSEC作为判断只有峰值部分应该合并，浓缩。 SERT-8B6晶体生长在一个窄的范围内的条件，并有许多的应采取解决具体涉及SERT晶体生长问题的步骤。 Tris碱用HCl调节不应在贮存溶液中使用，因为这种缓冲器不支持晶体生长;因此它是CRI蒂卡尔改用的Tris用NaOH调节。 SERT晶体生长在聚乙二醇400的窄的浓度范围内，因此，如果晶体不生长或如果观察到许多小晶体，即使在PEG 400浓度的小的增加或减少会被告知。此外，该添加剂6-氨基己酸也被在优化屏幕用于改善成核。的蛋白质的降低率:井溶液也是一个关键的决定因素为晶体生长。降的1.5比 - 2:1，建议，与降比率更接近2:1通常支持较大的三维晶体的生长。最后，使用低轮廓24孔板的也是朝向晶体增长的关键，这大概是由于蒸汽扩散的速率的修饰。 该SPA方法的另一种方法已经发展到屏幕的量稳定的大鼠血清素转运中使用的过滤器结合试验5可卡因结合构象突变。与此相反，对SPA巴SED法允许通过测定SERT的这仍结合配体部分的顺序如下加热步骤。从而，这允许从一个小数目的样本的快速测定熔融温度。 SPA的方法依赖于放射性标记的高亲和性配体的可用性，如果没有配位体是已知的具有亚微摩尔的亲和力结合然后一种替代方法将是必要的。许多其它方法通常用于测量蛋白质稳定性的诸如荧光染料和量热14的结合，但是低通量，均无法直接测量功能或需要大量的蛋白质。如果不能使用了SPA方法，一个可替换的高通量的方法是一种基于FSEC-热稳定性试验15（FSEC-TS），其中样品被加热之后剩余转运的级分的分离。 FSEC-TS是访问色谱行为和低聚状态的有用的方法，是APowerful互补工具，它可以沿着SPA的方法中。 各种常用的蛋白质表达系统的比较也被发现有利于对SERT表达16使用哺乳动物细胞和勿庸置疑，这是有可能为哺乳动物来源的许多蛋白质的情况。我们已经用于表达的方法已被定制为SERT但有可能易于适应。这有利于高水平的表达条件应通过改变表达，温度，病毒浓度和存在的组蛋白脱乙酰酶抑制剂的时候，如丁酸钠仔细鉴别。 我们一般倾向于在温和的长链洗涤剂亲和纯化如C12M连同CHS以复溶之前保留高亲和力配体结合。使用疏水性的吸收重建是一种温和的技术，该技术我们已经发现是有效的几个其他转运和受体。如果这不成功，透析，稀释，或者SEC除去具有高临界胶束浓度洗涤剂可使用17提供的抗原是在这类洗涤剂充分稳定。在没有合适的抗体被发现的情况下，我们几乎总能找到问题是由于功能或抗原变性损失在这种情况下，我们成功地执行新的免疫接种要特别注意蛋白质生物化学。最后，这与高亲和力结合的配体和抗体应当形成用于合理规划结晶实验的基础，并通过采取热变体的优点，人们可以通过改变不同洗涤剂的属性屏幕的条件范围更广。此外，在脂质中间相18结晶或使用bicelles 19应该总是被视为在微胶粒的替代结晶。 这些原理和方法可与一些modifi使用阳离子为许多其他跨膜蛋白这是难以表达，并从其他表达宿主纯化和将成为的高亲和性的药物靶的结构测定是特别有用的。

血清素转运蛋白（SERT）是有助于羟色胺的穿过细胞膜1传送一个完整的膜蛋白。 SERT属于家庭神经递质钠Symporters（NSSS），其中还包括多巴胺和去甲肾上腺素转运2。 SERT是广泛的处方抗抑郁和抗焦虑药，其作用是竞争性抑制血清素转运3的分子靶点。 SERT利用钠的积极有利的协同转运从突触间隙去除神经递质。血清素系统的广泛的表征已经表明，血清素代谢的变化看起来几乎影响所有神经过程，包括情绪，睡眠，疼痛，认知和攻击行为4。 SERT功能可以通过使用，比如s -citalopram，抗抑郁药和选择性血清素再摄取抑制剂（SSRIs），以及由psychostimula修改NTS和吸毒成瘾的药物，如安非他明和3,4-亚甲ñ-methylamphetamine或"摇头丸"1,2。 SSRIs的是心境障碍的治疗极为重要的，但对于其作用的确切结构基础还不是很清楚。 WT SERT是在去污剂胶束不稳定，因此妨碍朝SERT 5,6的三维（3D）结构的进展。最近，我们开发这是鲁棒稳定在广泛的洗涤剂和保留SSRI结合活性6 SERT的变体。这些热稳定的SERT变体使用基于邻近闪烁热稳定性测定法中选择。在这里，我们描述了可结合SERT高亲和力抗体的产生的方法，以及热稳定SERT的纯化和结晶，在用抗体和S -citalopram复杂。 这个协议假定SERT和8B6基因已经全成ý分别克隆入BacMam 7和昆虫表达载体。为了产生抗体，编码cDNA残WT SERT的73-616克隆到用C-末端链球菌II标签（SERT IC）的BacMam载体。对于热稳定性屏幕，使用SERT残基73-616具有C末端的GFP，链球菌II和10-His的标签（SERT TC）。在SERT TC背景生成个别的点突变。对于结晶协议中，SERT-GFP融合蛋白与双链球菌[TrpSerHisProGlnPheGluLys（GlyGlyGlySer）2 GlyGlySerAlaTrpSerHisProGlnPheGluLys]和10他的标签中使用，携带耐高温突变体，Y110A，I291A和T439S和表面半胱氨酸C554A，C580A突变和C622A（SERT CC）。凝血酶裂解序列（LVPRGS）也插入在Q76和T618后SERT CC以允许除去的N-和C-末端。编码8B6的Fab的质粒被工程化以表达两个重链和轻与两个单独的多角体蛋白启动子的控制下，GP67的分泌序列的抗体链。所述8B6抗体的重链的C-末端被标记使用8 His标签和被插入重链和标签之间的凝血酶切割位点。

<table><tbody><tr><td>DH10Bac</td><td>Invitrogen</td><td>10361-012</td><td></td></tr><tr><td>Kanamycin</td><td>Fisher</td><td>BP906-5</td><td></td></tr><tr><td>Gentamicin</td><td>Fisher</td><td>BP918-1</td><td></td></tr><tr><td>Tetracycline</td><td>Sigma</td><td>T-7660</td><td></td></tr><tr><td>Bluo-gal</td><td>Invitrogen</td><td>15519-028</td><td>5-Bromo-3-indolyl &amp;beta;-D-galactopyranoside</td></tr><tr><td>Isopropyl beta-D-1-thiogalactopyranoside</td><td>Anatrace</td><td>I1003</td><td>IPTG</td></tr><tr><td>Miniprep kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>Cellfectin II</td><td>Invitrogen</td><td>10362-100</td><td>Sf9 transfection reagent</td></tr><tr><td>Sf9</td><td>ATCC</td><td>CRL-1711</td><td></td></tr><tr><td>Sf-900 III SFM media</td><td>Life Technologies</td><td>12658-027</td><td></td></tr><tr><td>HEK-293S GnTI&lt;sup&gt;-&lt;/sup&gt;</td><td>ATCC</td><td>CRL-3022</td><td></td></tr><tr><td>Freestyle 293 media</td><td>Life Technologies</td><td>12338-018</td><td>293 expression media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>0984018DJ</td><td></td></tr><tr><td>Sodium butyrate</td><td>Sigma</td><td>303410</td><td></td></tr><tr><td>&lt;em&gt;S&lt;/em&gt;-citalopram</td><td>Sigma</td><td>E4786</td><td>Anagrade</td></tr><tr><td>n-Dodecyl-beta-D-Maltopyranoside</td><td>Anatrace</td><td>D310</td><td></td></tr><tr><td>Cholesteryl hemmisuccinate</td><td>Sigma</td><td>C6013</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine</td><td>Avanti Polar Lipids</td><td>850457P</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine</td><td>Avanti Polar Lipids</td><td>850757P</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoglycerol</td><td>Avanti Polar Lipids</td><td>840457P</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884</td><td></td></tr><tr><td>Pepstatin A</td><td>Sigma</td><td>P5318</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>Desthiobiotin</td><td>Iba Life Sciences</td><td>2-1000-05</td><td></td></tr><tr><td>Asolectin</td><td>Sigma</td><td>11145</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C-8667</td><td></td></tr><tr><td>Lipid A</td><td>Sigma</td><td>L5399</td><td></td></tr><tr><td>Brain polar lipid</td><td>Avanti Polar Lipids</td><td>141101C</td><td></td></tr><tr><td>Biobeads</td><td>Biorad</td><td>152-3920</td><td>Hydrophobic absorption resin</td></tr><tr><td>Goat anti-mouse IRDye 680RD</td><td>Odyssey</td><td>926-68070</td><td>Used as secondary antibody for western blotting</td></tr><tr><td>Lauryl maltose neopentyl glycol</td><td>Anatrace</td><td>NG310</td><td>Anagrade</td></tr><tr><td>Serotonin</td><td>Sigma</td><td>H9523</td><td></td></tr><tr><td>pFastBac 8B6</td><td></td><td></td><td>Available from authors</td></tr><tr><td>pEG Bacmam SERT Strep II</td><td></td><td></td><td>SERT&lt;sub&gt;IC&lt;/sub&gt;, Available from authors</td></tr><tr><td>pEG Bacmam SERT GFP twin Strep His</td><td></td><td></td><td>SERT&lt;sub&gt;TC&lt;/sub&gt;, Available from authors</td></tr><tr><td>pEG Bacmam SERT ts3 GFP twin Strep His</td><td></td><td></td><td>SERT&lt;sub&gt;CC&lt;/sub&gt;, Available from authors</td></tr><tr><td>Imidazole</td><td>Sigma</td><td>56749</td><td></td></tr><tr><td>n-Octyl &amp;beta;-D-maltoside</td><td>Anatrace</td><td>O310</td><td>Anagrade</td></tr><tr><td>Thrombin</td><td>Haematologic Technologies</td><td>HCT-0020</td><td></td></tr><tr><td>EndoH</td><td>New England Biolabs</td><td>P0702</td><td></td></tr><tr><td>Trizma-HCl</td><td>Sigma</td><td>T5941</td><td>Tris is used for preparation of crystallization reservoirs</td></tr><tr><td>PEG 400</td><td>Sigma</td><td>91893</td><td></td></tr><tr><td>6-aminohexanoic acid</td><td>Sigma</td><td>7260</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Fisher</td><td>MT25052CV</td><td></td></tr><tr><td>Isoplate-96 TC</td><td>PerkinElmer</td><td>6005070</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen</td><td>SL100688</td><td>Polymer transfection reagent for mamalian cells</td></tr><tr><td>Copper HIS-Tag YSI SPA Beads</td><td>PerkinElmer</td><td>RPNQ0096</td><td>His-tag affinity SPA beads</td></tr><tr><td>Citalopram, [N-Methyl-&lt;sup&gt;3&lt;/sup&gt;H]</td><td>PerkinElmer</td><td>NET1039250UC</td><td></td></tr><tr><td>ThermoMixer C</td><td>Eppendorf</td><td>5382000023</td><td>heating block for thermostability assay</td></tr><tr><td>ThermoTop</td><td>Eppendorf</td><td>5308000003</td><td></td></tr><tr><td>SmartBlock PCR 96, thermoblock for PCR plates</td><td>Eppendorf</td><td>5306000006</td><td></td></tr><tr><td>0.2 &amp;micro;m syringe filter</td><td>Olympus Plastics</td><td>25-243</td><td></td></tr><tr><td>1 L filter system</td><td>Corning</td><td>430517</td><td></td></tr><tr><td>2 L flat bottom tissue culture flask</td><td>Genemate</td><td>F-5909-2000</td><td></td></tr><tr><td>2 L baffled tissue culture flask</td><td>Genemate</td><td>F-5909-2000B</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Scientific</td><td>3950</td><td></td></tr><tr><td>Forma Orbital Shaker</td><td>Thermo Scientific</td><td>416</td><td></td></tr><tr><td>Strep-Tactin resin</td><td>Iba Life Sciences</td><td>2-1208-025</td><td>Strep affinity resin</td></tr><tr><td>Extruder</td><td>Northern Lipids</td><td></td><td></td></tr><tr><td>Li-Cor imaging system</td><td>Odyssey</td><td></td><td>western blot imaging system</td></tr><tr><td>XK16 column</td><td>GE Healthcare</td><td>28-9889-37</td><td>column used for Strep-Tactin and Talon purificaiton</td></tr><tr><td>100 kDa MWCO protein concentrator</td><td>Millipore</td><td>UFC910096</td><td></td></tr><tr><td>30 kDa MWCO protein concentrator</td><td>Millipore</td><td>UFC903024</td><td></td></tr><tr><td>&amp;Auml;kta FPLC</td><td>GE Healthcare</td><td>UPC-900</td><td></td></tr><tr><td>HPLC</td><td>Shimadzu</td><td>51476</td><td></td></tr><tr><td>Superose 6 (10/300) column</td><td>GE Healthcare</td><td>17-5172-01</td><td>Used for FSEC</td></tr><tr><td>Tangential flow apparatus</td><td>Pall Filtron</td><td></td><td></td></tr><tr><td>0.2 &amp;micro;m filter tangential flow cell</td><td>Pall Filtron</td><td>PSM20C11</td><td></td></tr><tr><td>30 kDa MWCO tangential flow concentrator</td><td>Pall Filtron</td><td>OS030T12</td><td></td></tr><tr><td>Talon resin</td><td>Clonetech</td><td>635504</td><td>His-tag affinity resin used for Fab purification</td></tr><tr><td>1 mL HiTrap SP column</td><td>GE Healthcare</td><td>17115101</td><td>Cation exchanger used for Fab purification</td></tr><tr><td>Superdex 200 10/300 GL column</td><td>GE Healthcare</td><td>17-5175-01</td><td>Used for SEC separation of SERT-8B6</td></tr><tr><td>24-well VDXm plate</td><td>Hampton Research</td><td>HR3-306</td><td></td></tr><tr><td>18 mm coverslips</td><td>Hampton Research</td><td>HR3-239</td><td></td></tr><tr><td>Virocyt virus counter</td><td>Virocyt</td><td>2100</td><td></td></tr><tr><td>MicroBeta Trilux</td><td>PerkinElmer</td><td>1450</td><td>96-well scintillation counter</td></tr><tr><td>HiTrap SP column</td><td>GE Healthcare</td><td>17115101</td><td></td></tr><tr><td>Sertraline</td><td>Sigma</td><td>S6319</td><td></td></tr></tbody></table>

thermostabilization, expression, purification, and crystallization of the human serotonin transporter bound to s-citalopram

羟色胺转运是钠和氯偶联转运的"泵"外血清素进入细胞:S -citalopram是用于通过结合具有高亲和力的羟色胺转运，阻断血清素再摄取来治疗抑郁症和焦虑症的药物。在这里，我们报告一个有效的程序和一套工具，以稳定，快速，纯化和结晶势必给S -citalopram和其他抗抑郁药羟色胺转运-抗体复合物。其中稳定羟色胺转运突变使用S -citalopram结合分析鉴定。在杆状病毒转导HEK293S GnTI表示羟色胺转运-细胞，被改组为脂酶和用于提高高亲和力的抗体。我们已经制定了一项战略，以发现对于结构研究的抗体。抗体片段在昆虫细胞中表达的直接方法也已确立。使用这种方法纯化运输车-抗体复合物的乖巧和容易结晶，产生带有S -citalopram的衍射X射线，以3-4的分辨率配合。这里开发的策略可以被用来确定其他具有挑战性的膜蛋白的结构。

在SERT TC背景的单点突变体库的建立是为了屏幕thermostabilizing突变。使用标准的诱变产生的变异个体。筛选协议利用瞬时转HEK293S细胞和基于接近闪烁热稳定性屏幕迅速身份进行结晶有用的突变如在图1A中所述。绘制的Tm值与结合的[3 H]西酞普兰在RT揭示具有适当高的热稳定性和表达水平构建蛋白质纯化（ 图1B）。三突变体（Y110A，I291A和T439S）合并以产生高度稳定的构建体（ 图1C）。热稳定性也与在必要的SERT-Fab复合的结晶化短链洗涤剂稳定性增加相关。 利用杆状病毒-TR人类SERT的大规模表达ansduced HEK293S GnTI -细胞可以少于2周，并且能够产生毫克量，如在图2A中示出。的GFP标记SERT CC蛋白的使用允许SERT通过荧光（ 图2B）的表达和纯化过程中方便地遵循。我们的纯化策略涉及1）SERT的增溶从HEK293S GnTI结合至S -citalopram -细胞C12M在CHS的存在作为稳定脂质; 2）SERT到链球菌亲和基质结合; 3）去除充分洗涤污染的蛋白质;和4）用含有缓冲液脱硫生物素（ 图2C）的官能SERT的溶出。洗脱的蛋白是基本上免费通过考马斯蓝染色等可检测的蛋白质和单分散由FSEC（ 图2D，E），来判断。 类似的策略被送往净化SERT与用于reconstitutio链球菌II标签n和免疫（ 图3A，B）。 SERT掺入脂酶提高SERT的血清半衰期和稳定性，并改善高亲和力抗体的隔离的可能性。此外，包含脂质A的，细菌的细胞壁的组分，用作有效的佐剂9。多层脂质体通过加入缓冲到玻璃试管中干燥的脂质混合物的制备和再悬浮于缓冲液中。通过200纳米孔径的过滤器脂质体的挤压产生单分散的单层脂质体悬浮液。脂质体，然后用洗涤剂随后在洗涤剂中加入纯化的SERT的饱和。洗涤剂混合物:最后，洗涤剂通过加入疏水性吸附树脂到脂质的除去。附加配位体应添加到重构的样本选择为识别的抗抑郁剂结合的构象的抗体。 SERT在脂蛋白存在应予以确认增溶用SDS-PAGE加样染料或C12M少量样品和在SDS-PAGE和FSEC（ 图3C，D）的运行。 表达SERT抗体的杂交瘤细胞系可筛选能识别三维表位的高亲和力结合物。这些性能对结晶的最终成功是至关重要的，因为抗体必须保持牢固地结合到结构化区域，以促进均匀，良好有序的域的晶体堆积。在第一步骤中，其识别非结构化区的抗体进行识别。 SERT变性并印迹到硝酸纤维素膜;它结合变性SERT抗体将成为西部阳性，有可能识别线性表位。在图4A中，我们显示抗体2其例子是西方阳性且可能不会有用，以促进crystallogenesis。在图4B中，剩余的西部阴性抗体孵育用100nM SERT-GFP和分隔FSEC。其结合SERT将GFP阳性峰转移至先前位置的抗体。的SERT - 抗体复合物可以在洗涤剂进一步稀释以确定它们是否可以与纳摩尔亲和力接着分析由FSEC结合。在转运的构象变化，从而抗体的血清素的结果的加法可以再次筛选以确定它们是否能特异性识别SSRI结合构象。在图4C中 ，抗体显示结合SERT在血清素的存在，显示出表位不脱离SSRI改变到衬底束缚态。最后，在图4D的抗体的组合被用于其结合不同的表位，从而产生一个进一步左移的能力。这里15B8或8A11抗体识别的表位是从8B6不同。 所述8B6抗体是基于用木瓜蛋白酶TREA初步结晶筛选选择用于进一步的结构分析特德晶圆厂。的8B6的Fab的基因被克隆到昆虫细胞表达载体。晶圆厂能够表达和从Sf9细胞悬浮生长分泌。的8B6的Fab可以从的Sf9细胞上清液通过His标签的亲和力（ 图5A，B）和得到的在其中出现自由在SDS-PAGE凝胶上的污染物的蛋白质的阳离子交换色谱法（ 图5C，D）的纯化。在图5E中，重组8B6的Fab被示出为结合SERT，并在随后的生化和生物物理实验时使用。 亲和纯化的SERT CC被消化凝血酶和EndoH的并与8B6的Fab混合以形成S中-citalopram的存在的复合物。转运-抗体复合物，然后通过SEC在C8M（ 图6A）分离，并且将峰值馏分同时包含SERT和Fab所示通过SDS-PAGE（ 图6B）。 C8M的使用结晶形成可能是因为关键短链洗涤剂允许在晶格分子之间更好的包装。 FSEC被用来确定哪个级分应该合并用于结晶（ 图6C）;馏分哪些不是单分散和/或含有大量游离SERT或Fab的不应该结合。 棱柱形SERT抗体晶体可以S中-citalopram的存在下，通过悬滴蒸气扩散（ 图7A）使用该协议来生长。所生成的晶体衍射X射线，以3.15 10（ 图7B）的分辨率。 <img alt="图1" src="/files/ftp_upload/54792/54792fig1.jpg" /> 图1:闪烁亲近基于热稳定性测定 采用 。协议用于在[3 H]西酞普兰。B存在下筛选热稳定性概述。最大约束[3 </SUP&gt; H]西酞普兰与表观熔融温度（Tm）。虚线表示的WT转运值。 3最耐热的突变体标记。灰色区域表示具有的[3 H]西酞普兰结合相对于WT和因而不准确的Tm值低于10％的突变体由于低信号对噪声。℃。对WT SERT TC和前3个突变体的热稳定性曲线。误差线表示标准偏差（SD）。 <a href="http://ecsource.jove.com/files/ftp_upload/54792/54792fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54792/54792fig2.jpg" /> 图2:哺乳动物异源蛋白表达的概述 。在HEK293S GnTI BacMam病毒的产生和SERT的表达图式概述-小区B。他K293S GnTI -表达SERT CC（绿色荧光）C细胞。上链球菌亲和树脂的SERT CC的洗脱图。绿色迹线表示脱硫生物素的浓度，0 - 100％（0 - 5毫米）D。分析4亲和纯化的SERT 的CC - 15％SDS-PAGE凝胶即 。通过GFP荧光检测亲和纯化SERT CC的FSEC（激发:480纳米;发射:510纳米）。高峰在15毫升洗脱是SERT（＃）和18毫升是免费的GFP（*）。 <a href="http://ecsource.jove.com/files/ftp_upload/54792/54792fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54792/54792fig3.jpg" /> 图3:在SERT IC A 的代表亲和纯化和重组 。抗体产生的示意图。 &lt;强&gt;乙。洗脱曲线在SERT 的IC上链球菌亲和树脂的亲和纯化的280nm处观察到。绿色迹线表示脱硫生物素的浓度，0 - 100％。（0 - 5毫米）℃。纯化，并在4重构SERT亲和力的分析- 15％SDS-PAGE凝胶ð。以下重建溶解SERT的FSEC。用色氨酸残基的荧光检测SERT（激发:280纳米;发射:335纳米）。 <a href="http://ecsource.jove.com/files/ftp_upload/54792/54792fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54792/54792fig4.jpg" /> 图4:代表SERT抗体的分析 的 。 Western blot检测抗体的筛选。约1微克有或没有GFP SERT 的CC被加到4 - 15％SDS-PAGE凝胶并印迹到硝酸纤维素膜上。结合使用缀合到IR染料山羊抗小鼠抗体检测。 2G4和10F2是西方积极的; B。抗体由FSEC 100纳米GFP标记SERT和检测的结合使用GFP荧光检测温度 。选定的Fab结合于100nM的GFP标记的SERT在1mM的血清素。D的存在。 8A11或15B8 Fab的结合SERT-8B6晶圆厂。小峰在18毫升洗脱是免费的GFP。 <a href="http://ecsource.jove.com/files/ftp_upload/54792/54792fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54792/54792fig5.jpg" /> 图5:从昆虫细胞的8B6的Fab代表 一个 净化。洗脱分布在8B6的Fab的纯化的280纳米His标签的亲和力chromatograp观察<html> HY。绿色迹线表示的咪唑的浓度，0 - 50％（0 - 250毫摩尔） 乙 。非还原和His标签亲和纯化后还原SDS-PAGE凝胶。它运行邻近50 kDa蛋白是非还原的Fab（＃），并在25 kDa的被减小的Fab（*）。C小调物种。洗脱曲线在8B6的Fab的纯化的280纳米通过显示该线性氯化钠梯度下洗脱的单个对称峰阳离子交换观察。绿色迹线表示NaCl浓度，0 - 100％。（0 - 500毫）D。以下通过阳离子交换的E纯化在12.5％SDS-PAGE凝胶的8B6的Fab的分析。在8B6的Fab结合纳米10 GFP标记的SERT，利用GFP的荧光检测。 <a href="http://ecsource.jove.com/files/ftp_upload/54792/54792fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> Ë6"SRC ="/文件/ ftp_upload / 54792 / 54792fig6.jpg"/&gt; 图6:S中的存在的-citalopram A 中的SERT-8B6复杂的代表凝胶过滤层析 。纯化SERT-8B6复杂的凝胶过滤洗脱图。主峰为11.5毫升洗脱是SERT-8B6复杂。峰15 - 17毫升含有绿色荧光蛋白和Fab 湾 。分析在4纯化的SERT-8B6络合物 - 15％SDS-PAGE凝胶。 SERT和Fab重链和轻链的位置由一个破折号，C中所示。尺寸分离级分的FSEC。利用色氨酸荧光检测SERT-8B6复合物。部分17包含没有用复杂的Fab SERT的用量较大。 <a href="http://ecsource.jove.com/files/ftp_upload/54792/54792fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7"SRC ="/文件/ ftp_upload / 54792 / 54792fig7.jpg"/&gt; 图7:在SERT-8B6复杂绑定至S -citalopram A 的结晶 。 2周增长后的SERT-8B6复杂的平行六面体状晶体的光镜。比例尺等于200微米; B。 SERT-8B6晶体衍射X射线到3.15埃。蓝环代表3.15埃。 <a href="http://ecsource.jove.com/files/ftp_upload/54792/54792fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 表 1:结晶屏幕为SERT-8B6复杂绑定至 S -citalopram <a href="http://ecsource.jove.com/files/ftp_upload/54792/54792table1.xlsx">请点击这里下载此表。</a>

Watch this Scientific Journal Video about Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram at JoVE.com

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

这份手稿介绍如何筛选thermostabilizing突变，净化人的羟色胺转运，产生高亲和力的抗体，并结晶势必抗抑郁药物小号 -citalopram羟色胺转运-抗体复合物。该协议可以适于其他具有挑战性的膜转运，受体和通道的研究。

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 ...

thermostabilization-expression-purification-crystallization-human

Research

JoVE Journal

Biochemistry

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

15.0K 次观看.  Oregon Health & Science University. 这份手稿介绍如何筛选thermostabilizing突变，净化人的羟色胺转运，产生高亲和力的抗体，并结晶势必抗抑郁药物小号 -citalopram羟色胺转运-抗体复合物。该协议可以适于其他具有挑战性的膜转运，受体和通道的研究。 

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

Expression, Purification, and Antimicrobial Activity of S100A12

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 S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

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 ...

科研

生物化学

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

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

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

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

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 ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

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 ...

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

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

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 ...

表达和纯化溶质载体转运蛋白的策略

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

作者聚焦:使用密码子优化基因表达和纯化人溶质载体转运蛋白 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

用于结构和生化研究的人溶质载体的高通量表达和纯化

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

免疫与感染

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

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

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

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到

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