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
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.).
Materials
| Culture Flasks | GeneMate | F-5909B | |
| 293 Freestyle Media | Gibco/Life Technologies | 12338-018 | |
| GlutaMAX | Gibco/Life Technologies | 35050-061 | Use in place of Glutamine |
| Hype 5 transfection reagent | Oz Biosciences | HY01500 | |
| 293fectin transfection reagent | Life Technologies | 12347019 | |
| PEI transfection reagent | Sigma-Aldrich | 408727 | |
| Maxiprep Kit | Qiagen | 12162 | |
| Ni-NTA Superflow | Qiagen | 30430 | |
| Endo Hf | NEB | P0703L | |
| Amylose Resin | NEB | E8021S | |
| Cell Boost R05.2 | HyClone | SH30584.02 | Cell Culture Supplement |
| GlucCell | CESCO Bioengineering | DG2032 | Glucose Monitoring System |
| Opti-MEM | Life Technologies | 519850.91 | Serum Free Medium for DNA transfection |
| Luria Broth (LB Media) | Life Technologies | 10855-001 | |
| GC10 Competent Cells | Sigma-Aldrich | G2919 |
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