Name: efficient mammalian cell expression and single-step purification of extracellular glycoproteins for crystallization
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This work was supported by NIH R01-HL119813 (to T.J.B.), American Lung Association RG-196051 (to T.J.B.), a CIMED Pilot and Feasibility grant (to T.J.B.), American Heart Association Predoctoral Fellowships 14PRE19970008 (to Z.Y.) and 15PRE22110004 (to D.L.K.).

1.生产质粒DNA的毫克量为大规模瞬时转染的<ol><li>克隆感兴趣的蛋白质成使用限制性位点克隆，或其它适当的技术高的拷贝数的哺乳动物表达载体中。 <ol><li>为获得最佳的结果，使用pHLsec 7矢量，其中有一个内置的C末端的6His标签，强启动子Kozak序列和优化的分泌信号。 </li></ol></li><li>变换质粒到感受态细胞。 <ol><li>添加20微升感受态大肠杆菌细胞的到1微克质粒DNA和在冰上孵育30分钟。 </li><li>热休克的细胞在42℃下35秒，然后在冰上孵育2分钟。 </li><li>添加300μl的微生物生长培养基（SOC），并在37℃下进行45分钟，摇动以220rpm。 </li><li>板细胞在琼脂平板上适当的抗生素选择。 <ol><li>使用100微克/毫升羧苄青霉素，如果所述质粒是在pHLsec矢量。 </li></ol></li></ol></li><li>在250ml的Luria肉汤（LB）媒体的培养菌落补充有100微克/毫升抗生素（羧苄青霉素）O / N在37℃下，振荡以220rpm。 </li><li>净化从根据制造商的方案使用高速质粒Maxi试剂盒培养物DNA。 <ol><li>洗脱，代替缓冲器的TE缓冲液中的EB（10mM的三Cl，pH值8.5）的DNA。 </li><li>分装纯化的质粒在在-20℃所需的转染和存储量。 </li></ol></li></ol>的293F细胞2.大型文化和瞬时转染<ol><li>补充的1L 293F媒体用10ml谷氨酰胺和5ml青霉素/链霉素（均100倍）。保存在4℃。 5 ml的青霉素/链霉素是一个足够的强度在无血清条件下转染和在减压抗生素浓度提高细胞生存力，从而提高蛋白质的产量。 </li><li>文化293F细胞在300毫升的媒体在1升的聚碳酸酯挡板锥形瓶中通气帽s的37℃，8％CO 2中，一边摇动在标准组织培养箱中。 </li><li>稀释细胞之一转染前一天5×10 5 / ml的密度。 </li><li>转染，补充培养基中加入的2％10％体积w / v的在293F媒体电池升压的一天。 <ol><li>使用根据制造商的说明和使用补充剂根据需要以达到500毫克/分升的葡萄糖浓度的血糖监测仪测量葡萄糖浓度。 </li></ol></li><li>加kifunensine（1微克/毫升的最终浓度）在此步骤中，以控制蛋白质的糖基化。 </li><li>的DNA的计算量所需的每1×10 6个细胞1微克质粒。在无菌条件下，稀释在5ml无血清培养基中的DNA。 </li><li>计算所需的每2 1微克质粒微升转染试剂转染试剂的体积。在无菌条件下，稀转染试剂（PEI-TMC-25）在5ml无血清培养基。 </li><li>加转染试剂成在1ml的增量DNA溶液，轻轻混合。温育30分钟，在RT下试剂-DNA复合物的形成。然后将溶液添加到细胞中逐滴的方式。 </li><li>允许转染的细胞表达蛋白72-96小时。补充用〜10％的体积电池升压日常必要的媒体，或保持血糖读数400〜600毫克/分升。 </li></ol> 3.净化<ol><li>滗培养到离心瓶中，离心20分钟，1300×g离心以沉淀细胞，然后回收上清。如果需要的话，旋转的第二时间和/或使用0.22μm的过滤器，以澄清上清液。 </li><li>加10％的体积的10倍的Ni-NTA结合缓冲液（1.5M氯化钠，0.5 MK 2 HPO 4，0.1M的Tris pH值8.5，50mM咪唑）。 </li><li>通过加入2ml的Ni-NTA浆在一个柱并用1X结合缓冲液10倍柱体积（CV）的平衡准备一个重力柱。如果可能的话，请在4℃的房间内的所有列的步骤。可选择性伊利，寒意蛋白和列步骤之前，冰所有的缓冲区，并保持蛋白质和收集流过冰。 注意:的Ni-NTA浆料是50％的树脂体积和制造商的说明结合容量为50毫克/毫升。的Ni-NTA珠可以被重新充电用于多种用途的</li><li>流上清液过树脂，并收集流过。重复此步骤。 </li><li>用10CV的洗涤缓冲液洗涤（300毫摩尔NaCl，50 mM的K 2 HPO 4，20mM咪唑的pH 8）中。 </li><li>洗脱蛋白在5 CV的洗脱缓冲液（300mM NaCl的50 mM的K 2 HPO 4，250mM咪唑pH 8）中。 </li><li>如果去糖基化是必需的: <ol><li>为0.5 ml的终体积，洗脱液浓缩，用离心浓缩器0.43毫升如果沉淀的形式，通过离心沉淀的任何碎屑在16000×g离心和4℃。 </li><li>加入50微升500毫米柠檬酸钠pH值5.5。 </li><li>加入20微升EndoHf（1×10 6单位/毫升）中。在室温下孵育2小时。 不E:酶工作最佳，在37℃，这可能会导致浓缩的蛋白质聚集，延长对RT温育后，如果去糖基化，通过SDS-PAGE或免疫印迹评估，是不完整的。酶不具有在4℃下的活性。 </li><li>以除去EndoHf:洗净直链淀粉树脂3倍在磷酸盐缓冲盐水（PBS）或最终贮存缓冲液。孵育蛋白质与树脂1小时，在4℃。自旋5分钟，在1000×g离心沉淀珠粒并回收上清。 </li><li>使用适当的分子量截留离心过滤器浓缩蛋白质和缓冲液交换成存储缓冲液（150毫摩尔NaCl和20mM HEPES pH7.5）中。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

HEK 293F细胞提供强大的生产需要翻译后修饰的蛋白质。该系统允许含有二硫化物，糖基化，和磷酸化，否则将使用更常规的表达的工具不存在本机折叠的蛋白质的快速且可伸缩的表达。此外，该系统可用于表达和多蛋白复合物的纯化只需通过共转染多种质粒的构建。此外TREM2，该系统已经被广泛地用于与感兴趣在实验室-10,11-其它蛋白质功能研究。哺乳动物细胞还提供用于体内实验或生产的原生折叠抗原以产生构象依赖性抗体无内 ​​毒素蛋白表达最佳。 最优细胞活力和转染条件是有效的蛋白生产的最关键的。为了获得更好的细胞生存力，低传代细胞培养物被提升用细胞培养物补充剂和生长培养基中的抗生素浓度降低。修改后的PEI-TMC-25具有与大多数蛋白质适合横断效率;但是，也有能提供增加的转染效率和减少的细胞毒性可用其他地点适中的价格。宣传5（盎司Bioscience公司）增加的产量相比PEI或其他试剂，在一个显着减少的成本相比，更昂贵的转染试剂，如的293fectin。试剂种类和DNA:试剂比可以为个别表达式和所希望的实验的需要进行优化。 这里所描述的方法具有超过蛋白质生产的其它方法有几个优点。哺乳动物细胞提供本地伴侣折叠和不可用的细菌和酵母的翻译后修饰。质粒构建和制备，沿着利用媒体的时间缩短，其可以直接修改的pH值，使之优于baculovir总部位于美国的生产在昆虫细胞;最后，在无血清培养基提供容易的缩放（更高的细胞密度）和纯化无法达到贴壁293T细胞。这个系统的主要缺点是仅在时间和缩放个人研究者就可以提交给蛋白的表达。 包括以下是遇到的最常见的问题进行故障排除的选项。 故障排除如果有低蛋白的表达，避免传代293F细胞比2-3个月的时间。长期传代将会降低细胞的健康体现在较慢的倍增时间和显著减少蛋白的表达。细胞生长应每天监测和文化每日不再加倍应被丢弃。细胞生存力可以通过使用培养补充剂而传代细胞得到改善。监测血糖，以确保400-600毫克文化/ dl浓度细胞密度不应超过2万/ ml和细胞活力应≥98％台盼蓝排除。稀释密集的细胞到0.5万/ ml，并允许双O / N前转。脱氧核糖核酸（1微克的DNA的范围内::1.5-6微升试剂），使用不同比例的转染试剂的转染优化。这可以通过使用2 ml的培养物在6孔板中并通过免疫印迹测定的输出来完成。做12小时的时间点可以指示如果蛋白质被表达，并迅速降解;检查细胞裂解物的蛋白质如果未在上清液部分观察。错折叠蛋白，它不能被分泌，仍然会在细胞裂解物通过免疫印迹显而易见。 如果的Ni-NTA保持为低， 即，蛋白质保留在流过柱结合后，用新鲜的树脂。 pH增加样本到8.5，将导致更强的结合到树脂上，虽然这也将增加非特异性结合。浓缩的样品和批量结合树脂O / N在4℃下前的Ni-NTA纯化可能导致〜5倍增加回收的蛋白质。如果的6His-tag为不可访问由于该蛋白质的三级结构，尝试在另一个末端克隆它。或者，使用钴树脂，这对他的残基的高亲和力。 如果蛋白聚集和/或纯化过程中沉淀，通过加入10％甘油成的Ni-NTA洗和洗脱缓冲液增加溶解度。运行镍亲和柱在室温而不是4℃。使用差示扫描荧光12以筛选pH和盐/添加剂增加在溶液中的蛋白质的稳定性。

了解蛋白质结构在原子级别是关键揭露生物学途径和疾病的分子基础。 X射线蛋白质晶体是用于确定大分子结构最广泛使用的/适用的方法。该方法的主要挑战是获得足够数量的正确折叠的，纯的蛋白质。这变得尤其分泌哺乳动物蛋白质，其经历特定的翻译后修饰工作时的一个问题。 细菌表达的蛋白质是沉积在蛋白质数据银行1中结晶蛋白质的主要来源。细菌表达系统在很大程度上是优选的，因为它们是快速，廉价和通常产生蛋白质的产量高。然而，哺乳动物蛋白的胞外域在细菌中表达通常不正确折叠的，在所要求的情况下的重折叠和大量的纯化步骤以获得均匀FOLDED蛋白质。此外，许多哺乳动物蛋白质需要的翻译后糖基化，以达到适当的折叠2。虽然在酵母或昆虫细胞中表达和糖基化可以克服的折叠问题，翻译后修饰，包括糖基化，显著从那些哺乳动物细胞3的不同，产生具有不正确的或不均质的修饰蛋白质。 哺乳动物细胞表达的所有必需的分子机制，以确保适当的翻译后修饰和折叠;然而，这些表达系统通常不优选由大多数实验室，由于有限的产率和试剂和消耗品成本高。聚乙烯亚胺（PEI），一个标准的转染试剂是相对便宜的，但施加相当的细胞毒性和低转染效率，从而导致在细胞培养基，脱氧核糖核酸成本增加，并培养设备。许多替代PEI是贵得离谱。我们解决这些问题通过描述和化学修饰的PEI的快速和相对便宜的方法为分泌的哺乳动物蛋白质的表达，随后单步纯化的改进的细胞培养工具的组合。这种强大的方法给出了足够的产率的功能和生化研究4中，在某些情况下，导致蛋白质适合于结晶，无需进一步纯化。 本协议描述的几种技术，以最大限度地表达和产生于人类胚胎肾（HEK）分泌的哺乳动物蛋白悬浮培养细胞293F。转染效率（成本），蛋白生产和纯化都极大地按照该协议增强。 PEI通过单步开环反应中加入氨基甲酸酯改性（PEI-TMC-25，合成和性质的详细在文献5中所述 ）大大提高转染效率，从阳离子减少的细胞毒性膜破坏，并相应地降低实验成本。此外，细胞存活率和蛋白质表达大大用加入了培养补充供应葡萄糖和维生素改善。重要的是，用于生产糖基化蛋白，治疗kifunensine的，甘露糖苷酶I的无毒的化学抑制剂，产生的蛋白质与定义，未成熟的聚糖，其可以通过该糖苷内切酶EndoHf被除去，得到的蛋白质与单个N-乙酰葡糖胺代替全长N-连接多糖6。最后，蛋白质分泌到无血清，化学定义的培养基允许快速和简便的纯化用于结构和生化研究。单步镍 - 次氮基三乙酸（的Ni-NTA）树脂纯化去除大多数在上清液污染物种类和，在某些情况下，可以产生足够的纯度为蛋白质结晶。

<table><tbody><tr><td>Culture Flasks</td><td>GeneMate</td><td>F-5909B</td><td></td></tr><tr><td>293 Freestyle Media</td><td>Gibco/Life Technologies</td><td>12338-018</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco/Life Technologies</td><td>35050-061</td><td>Use in place of Glutamine</td></tr><tr><td>Hype 5 transfection reagent</td><td>Oz Biosciences</td><td>HY01500</td><td></td></tr><tr><td>293fectin transfection reagent</td><td>Life Technologies</td><td>12347019</td><td></td></tr><tr><td>PEI transfection reagent</td><td>Sigma-Aldrich</td><td>408727</td><td></td></tr><tr><td>Maxiprep Kit</td><td>Qiagen</td><td>12162</td><td></td></tr><tr><td>Ni-NTA Superflow&amp;nbsp;</td><td>Qiagen</td><td>30430</td><td></td></tr><tr><td>Endo Hf</td><td>NEB</td><td>P0703L</td><td></td></tr><tr><td>Amylose Resin</td><td>NEB</td><td>E8021S</td><td></td></tr><tr><td>Cell Boost R05.2</td><td>HyClone</td><td>SH30584.02</td><td>Cell Culture Supplement</td></tr><tr><td>GlucCell</td><td>CESCO Bioengineering</td><td>DG2032</td><td>Glucose Monitoring System</td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>519850.91</td><td>Serum Free Medium for DNA transfection</td></tr><tr><td>Luria Broth (LB Media)</td><td>Life Technologies</td><td>10855-001</td><td></td></tr><tr><td>GC10 Competent Cells</td><td>Sigma-Aldrich</td><td>G2919</td><td></td></tr></tbody></table>

efficient mammalian cell expression and single-step purification of extracellular glycoproteins for crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

本文如下施加到分泌13 kDa的免疫球蛋白（Ig）的结构域，从表达的髓样细胞上2（hTREM2，残基19-132）的人蛋白质触发受体该表达系统的结果。 TREM2是包含具有两个二硫键和两个N-连接糖基化位点的胞外单个Ig结构域I型跨膜蛋白。不像许多其他的Ig结构域蛋白8，TREM2是不适合细菌包涵体9复性。随后的诱变证实N联聚糖都需要适当的表达和折叠。以促进结构和功能研究，TREM2引入pHLsec矢量与使用标准的分子生物学技术的C-末端的6His标签7。瞬时表达与kifunensine处理HEK293F细胞产生，其通过的Ni-NTA色谱法纯化蛋白（图1B，泳道2）和样品然后被去糖基化，以产生NATIvely折叠的，均匀的蛋白（图1B，泳道3）。 293F TREM2的表达产生5-10毫克/升（5-10μg/百万转染的细胞）。后缓冲交换到存储缓冲器中，该蛋白质中结晶（图1C）。尽管约80％的最终纯度，这些晶体可靠地再现，衍射，并分别所示银染色，以只包含TREM2蛋白（图1D）。这一观察表明该蛋白质的生化同质（即，折叠和翻译后修饰）可以，在某些情况下，比对结晶成功总纯度更为关键。 除了结晶，该系统提供了强大的工具，结构和功能的研究。它被利用，以产生糖基化的原生蛋白和达到&gt; 95％纯度，大小排阻色谱（图1E中的F）。这个额外的纯化步骤，其示出了搜索解决方案该蛋白质的n个行为，也监测蛋白质量的理想方式。将纯化的蛋白质应洗脱在对应于它的分子量的体积，并应是最丰富的种类与样品中。异常大量聚集蛋白质可能表明不稳定的蛋白质和提供线索，以优化蛋白的生产和纯化。此外，这个最后的纯化的蛋白是优良用于在定量生物物理实验使用如圆二色光谱，热稳定性，和调查蛋白质 - 配体相互作用。最后，控制糖基化程度提供了一种强大的工具，研究聚糖有关的功能。 <img alt="图1" src="/files/ftp_upload/53445/53445fig1.jpg" /> 图1.真核表达系统:（A）方案的蛋白质与HEK 293F细胞控制或天然糖基化哺乳动物表达。 （<html> B）的前（泳道2）中所示kifunensine和后（泳道3）去糖基化与EndoHf的SDS-PAGE NiNTA纯化hTREM2的表达。 （C）晶体从NiNTA纯化和去糖基化蛋白质产生的。结晶输入（D）的银染（泳道1）和收获晶体（泳道2和3）揭示了晶体只包含TREM2。 TREM2（E）的进一步纯化使用不Kifunensine在293F细胞中产生的NiNTA纯化TREM2的大小排阻层析来实现。星号（*）表示分子量标准的洗脱体积。蛋白质，使用分析S200柱（GE）洗脱在TBS中。 （F）的尺寸排阻纯化TREM2的SDS-PAGE分析:输入蛋白（泳道2）和折叠的峰（泳道3）。注意如何充分，与通过SDS-PAGE的广泛涂抹运行在更高的表观分子量异质聚糖。NK"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization at JoVE.com

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

efficient-mammalian-cell-expression-single-step-purification

Universidad San Sebastian

Research

JoVE Journal

Biology

13.7K 次观看.  Washington University School of Medicine. This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

视频: Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

科研

生物化学

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

哺乳动物 Bestrophin 离子通道的表达与纯化

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

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 ...

脂质双细胞中 ABC 转运蛋白膜蛋白的结晶

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

作者聚焦：用于 ABC 转运蛋白转运蛋白的 Bicelle 结晶设置以推进药物开发 

This protocol describes a setup for the crystallization of the sterol transporter ABCG5/G8. ABCG5/G8 is reconstituted into bicelles for hanging-drop crystallization. The protocol does not require specialized materials or substrates, making it accessible and easy to adapt in any laboratory for determining the protein structure through X-ray crystallography.

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an ...

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, ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

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用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

哺乳动物 Bestrophin 离子通道的表达与纯化

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

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

用于结构和生化研究的人溶质载体的高通量表达和纯化

用于结构和生化研究的人溶质载体的高通量表达和纯化

用于结构和生化研究的人溶质载体的高通量表达和纯化

一个从人体细胞分泌蛋白生产的方便，一般的表达平台