重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达
Summary
Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.
Abstract
产生重组蛋白的复杂的翻译后修饰的技术代表的结构和功能研究的主要挑战。迅速建立和高回收率从瞬时转染的哺乳动物细胞系解决了这一障碍并为表达该通过ER和高尔基体介导的分泌途径自然引导蛋白质的一种有效手段。这里是使用人类的HEK293F和转染的设计的高蛋白质产量的哺乳动物表达载体HEK293S细胞系表达一个协议。这个系统的适用性用与之间95-120毫克每升培养物中回收纯化的蛋白的产量表达三个具有代表性的糖蛋白是证明。这些蛋白质是人类和FcγRIIIa的大鼠α2-6唾液酸转移酶，ST6GalI，既表达具有N-末端GFP融合，以及未经修饰的人免疫球蛋白G1的Fc。这个强大的系统UTIlizes一个无血清培养基是能适应于同位素富集蛋白质和碳水化合物用于使用质谱和核磁共振谱结构研究的表达。此外，所述N-聚糖的组合物可以通过添加一个小分子，以防止某些聚糖修饰的方式，并没有降低产量来调节。
Introduction
制备适当折叠和翻译后修饰的人蛋白质的高产量的结构和功能的详细的分析仍然是一个显著挑战。可产生重组蛋白具有天然样的功能和行为的大量表达系统。细菌表达系统，主要是大肠杆菌菌株，代表了最方便和常用的工具，在研究领域，由于这些表达系统的简单性，尽管酵母，植物，昆虫和哺乳动物系统也描述1-4。然而，大多数这些系统是不能的靶蛋白的适当的翻译后修饰。锋芒毕露和Moremen实验室的根本利益，是生产真核蛋白与适当的糖基化。许多人类蛋白质需要适当的糖基化的功能是否正常（见图5）。
真核糖基化机制是广泛的，能够进行修改多种多样，包括天冬酰胺（N）的-和丝氨酸/苏氨酸（O） -连接的复合聚糖6。据估计，人蛋白质的> 50％是N-糖基化的7。聚糖是许多蛋白质包括治疗性单克隆抗体，促红细胞生成素，和凝血因子样因子IX的必要成分，仅举几例。虽然多种方法可用来制备适当N-糖基化蛋白和范围从纯合成8-10，以酶法11月14日或恢复从工程改造的重组系统15-20，这并不奇怪，人表达系统迄今被证明是最稳健的方法产生的人类蛋白质。
许多治疗人糖蛋白产生在使用哺乳动物细胞的重组系统。值得注意的系统是中国仓鼠卵巢（CHO），小鼠骨髓瘤（NS0），幼仓鼠KidneY（BHK），人胚肾（HEK-293），并且被用在粘附或悬浮培养生产蛋白质4,21,22人视网膜细胞系。然而，哺乳动物蛋白表达系统需要的稳定细胞株，昂贵的生长培养基和基质辅助转程序23的生成。
哺乳动物细胞的转染与众多试剂，包括磷酸钙24,25，阳离子聚合物（DEAE-葡聚糖，聚凝胺，聚赖氨酸，聚乙酰亚胺（PEI））或带正电荷的阳离子脂质体26-29的帮助来实现。 PEI是聚阳离子，充电，直链或支链的聚合物（25 kDa）的26，其形成稳定的络合物与DNA和被内吞。在核内体的酸化，PEI被认为是膨胀，导致内体和DNA的释放的破裂进入细胞质26,30。
直到最近，在suspensi瞬时转染上培养进行事先的DNA / PEI复合物的形成，随后加入到细胞培养物29。然而，亚龙和同事报道了生产重组蛋白的HEK293细胞在原位形成31,32的DNA / PEI复合物进行了优化的高效的协议。这避免了准备，复杂的灭菌，并进入培养基缓冲液交换。通过包括导致显著的产量增加33表达增强质粒进一步优化。这里是建立在这些进展和广泛适用的方法。表达的条件也可以被改变以影响N-聚糖组合物。
所述HEK293S细胞系，与基因缺失，其停止N-聚糖处理在中间级，导致与均匀N-聚糖的蛋白质组成的2 N-乙酰葡糖胺残基的加五甘露糖残基（曼5的GlcNAc 2）34中的表达， 35。这些细胞缺乏N-乙酰转移酶I（GntI），这是需要的下游N-聚糖处理36,37基因。使用糖基转移酶抑制剂，包括kifunensine，唾液酸类似物和岩藻糖类似物和2-脱氧-2-氟-岩藻糖也有类似的影响和限制N-聚糖处理38-41。
协议报这里使用pGEn2矢量如图1 42,43，PEI辅助瞬时转染到哺乳动物细胞系（HEK293F或HEK293S细胞），并适当地糖基化蛋白的高产率的恢复。这个系统是稳健，可容纳各种因素，包括同位素标记和聚糖工程用于生产重组蛋白的大效价。
Protocol
该协议是充分利用两种HEK293F或HEK293S细胞中的表达。 1.细胞建立培接种注：所有培养操作过程必须在BSL-2设施内进行和每个项目带入生物安全柜必须通过在水溶液中的70％的乙醇喷雾进行灭菌。 操作培养摇床中于135转，湿度80％，并在8.0％的CO 2和37℃。打开“的”生物安全柜中至少1小时前工作的紫外灯。 Prewarm密封培养基A和培养基B瓶在水浴在37℃进行1小时。 …
Representative Results
高水平表达和纯度这种优化的表达系统产生的高收率糖基化蛋白。典型的图案显示在IgG1的-Fc的（图1）的表达。在这种情况下，第0天是转染一天随后1天（稀释）和随后的培养天至第5天的蛋白质表达使用在粗培养基中的可溶性表达馏分进行分析。观察到在第1天的一个非常小的量的蛋白质表达作为培养等分试样取出?…
Discussion
这个协议说明通过HEK293F或S细胞的瞬时转染的蛋白表达。建立的倒钩和Moremen实验室的最佳转染条件采用细胞密度和试剂浓度的临界组合，以实现高效率的转染。实施这项协议时，关键的考虑因素包括：保持在转染前一个稳定的文化（具有一致的文化倍增时间）;活跃生长的细胞的转染细胞存活率大于95％（按稀释细胞至1×10 6个细胞/ ml 24小时在转染之前实现）;在转染细胞密度应在含90％培养…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由财政拨款K22AI099165（AWB）的支持，P41GM103390（KWM）和P01GM107012（KWM）由美国国立卫生研究院，以及来自生物化学，生物物理学和分子生物学的罗伊J.卡佛系美国爱荷华州立大学基金。这项工作的内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。
Materials
| Biosafety cabinet | NuAire, Inc. | CellGard ES NU-S475-400 | Class II, Type A2 Biological Safety Cabinet |
| Incubation shaker | INFORS HT | Multitron Cell | |
| Medium A: FreeStyle Expression Medium | Life Technologies | 12338-018 | |
| Medium B: ExCell 293 Serum-Free Medium | SIGMA | 14571C | |
| 125 Erlenmeyer Flask with Vented Cap | Corning Incorporated/Life Sciences | 431143 | |
| 250 Erlenmeyer Flask with Vented Cap | Corning Incorporated/Life Sciences | 431144 | |
| FreeStyle HEK 293F Cells | Life Technologies | R790-07 | |
| 1 ml Disposable serological pipette | Fisher Scietific | 13-676-10B | |
| 10 ml Disposable serological pipette | Fisher Scietific | 13-676-10J | |
| 25 ml Disposable serological pipette | Fisher Scietific | 13-676-10K | |
| Pipettor (Pipet-Aid XP) | Drummond Scientific | 161263 | |
| Trypan Blue Solution | Thermo Scientific | SV30084.01 | |
| Counting slides | Bio-Rad | 145-0011 | |
| TC20 Automated Cell Counter | Bio-Rad | 145-0102 | |
| Polyethylenimine (PEI) | Polysciences Inc. | 23966 | Prepare stock solution at a concentration of 1 mg/ml in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 150 mM NaCl (pH 7.5). Dissolve PEI completely; sterilize through 0.22 μM syringe filter and store at -20 °C. |
| 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | EMD Chemicals Inc. | 7365-45-9 | |
| 25 mm Syringe Filter, 0.22 μM | Fisher Scietific | 09-719A | |
| Trisaminomethane (Tris base) | Fisher Scietific | BP152-1 | |
| XL1-Blue | Stratagene | 222249 | |
| Trypton | Fisher Scietific | BP1421-2 | |
| Yeast extract | Fluka Analytical | 92144 | |
| Sodium chloride | BDH chemicals | BDH8014 | |
| Plasmid Purification Kit | QIAGEN | 12145 | |
| Valproic (VPA) | SIGMA | P4543 | Prepare stock solution of 220 mM in water, sterilize by passage through a sterile 0.22 mm filter and store at -20 °C |
| Corning 250 mL Centrifuge Tube | Corning Incorporated/Life Sciences | 430776 | |
| Centrifuge | Thermo Scientific | EW-17707-65 | |
| Protein A-Sepharose column | SIGMA | P9424 | |
| Ni-NTA superflow | QIAGEN | 30430 | |
| 3-(N-morpholino)propanesulfonic acid (MOPS) | Fisher Scietific | BP308-500 | |
| Glycine | Fisher Scietific | BP381-500 | |
| 10 kDa molecular weight cut-off Amicon® Ultra centrifugal filters | Millipore | UFC901096 | |
| Sodium dodecyl sulfate | ALDRICH | L3771 | |
| Beta-mercaptoethanol | ALDRICH | M6250 | |
| Glycerol | SIGMA | G5516 | |
| Precision Plus Protein All Blue Standards | Bio-Rad | 161-0373 | |
| Acetic Acid, Glacial | Fisher Scietific | 64-19-7 | |
| coomassie brilliant blue | Bio-Rad | 161-0406 | |
| MALDI-TOFMS Voyager-DE PRO | Applied Biosystems | ||
| 15N labeled L-Tyrosine | ALDRICH | 332151 | |
| 15N labeled L-Lysine | ALDRICH | 592900 | |
| Unlabeled L-Phenylalanine | SIGMA-ALDRICH | P2126 | |
| 13C6-Glucose | ALDRICH | 389374 | |
| 2-deoxy-2-fluoro-l-fucose | SANTA CRUZ Biotechnology | sc-283123 |
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Cite This Article
Subedi, G. P., Johnson, R. W., Moniz, H. A., Moremen, K. W., Barb, A. W. High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension. J. Vis. Exp. (106), e53568, doi:10.3791/53568 (2015).