完全加工重组KRAS4b：分离和描述脱脂和甲基化蛋白
Summary
前膜化是外周膜结合蛋白的重要修饰。昆虫细胞可以纵产生脱脂和甲苯甲酸化 KRAS4b 的数量，使生物物理测量蛋白质 – 蛋白质和蛋白质 – 脂质相互作用
Abstract
蛋白质前化是一种关键的修饰，负责将蛋白质定向到细胞内膜上。KRAS4b在22%的人类癌症中发生变异，由于C-总站存在”CAAX”箱形图案，KRAS4b通过脱氧核化和甲酸甲基化进行处理。一个工程的巴库病毒系统用于在昆虫细胞中表达脱脂和甲苯甲酸KRAS4b，并曾对此进行过描述。在这里，我们描述了蛋白质的详细，实用的纯化和生化表征。具体来说，亲和和电子交换色谱法用于纯化蛋白质，使蛋白质均匀化。使用正原质谱法验证KRAS4b的正确修改，并验证核苷酸结合。最后，利用表面质子共振光谱法，测定了脱氧核酸和甲苯甲酸化KRAS4b与脂质体之间的膜结。
Introduction
翻译后修饰在定义蛋白质的功能活性方面起着关键作用。磷酸化和糖基化等修改已经确立。然而，脂质修饰的特征较差。据估计，多达0.5%的细胞蛋白可能是1。前化是转移15碳法内西或20碳的Geranyl高脂链到含有CAAX图案2的接受蛋白。早发蛋白与多种人类疾病的进展有关，包括早衰3、阿尔茨海默氏症4、心脏功能障碍5、胆汁血症6和癌症7。小型GTPases、HRAS、NRAS和KRAS1、核层压宁和运动性CENP-E和F在基础条件下是远血化蛋白。其他小型GTPases，即RhoA，RhoC，Rac1，cdc-42和RRAS是Geranylranylate8，而RhoB可以是远发或杰兰格尔尼酸盐9。
小型GTPase KRAS4b功能作为分子开关，基本上通过多种蛋白质-蛋白质相互作用将细胞外生长因子信号传送到细胞内信号转导通路，刺激细胞生长和增殖。KRAS4b 生物化学有两个关键方面对其活性至关重要。首先，蛋白质在非活性GDP和活性GTP约束状态之间循环，从而主动与效应者互动。其次，C端多流氨酸区域和脱脂和甲苯甲基化半胱氨酸将蛋白质定向到血浆膜，使下游效应器得以招募和激活。突变KRAS4b是胰腺癌、结肠直肠癌和肺癌10的致癌驱动因素，因此，治疗干预将具有巨大的临床效益。生产真正改性重组蛋白，即脱脂和甲苯甲基化，可利用KRAS4b与脂质体或脂质纳米片等膜代用品进行生化筛选。
法内西尔转移酶 （FNT） 催化在 KRAS4b 的 CAAX 主题中向 C 端半胱氨酸中添加法尼西尔焦磷酸酯。前化后，蛋白质被贩运到内质神经质 （ER），其中 Ras 转换酶 （RCE1） 会裂解三个 C 端残留物。加工的最后一步是通过ER膜蛋白，异丙烯酰乙基甲基转移酶（ICMT）对新的C端法内西锡化残留物进行甲基化。大肠杆菌中重组KRAS4b的表达导致未修饰蛋白的产生。以前生产加工的KRAS4b的尝试一直受到限制，因为没有足够的产量的结构或药物筛选实验或未能重述本地全长成熟蛋白13，14。这里提出的协议采用基于毒细胞的工程性昆虫细胞表达系统和纯化方法，在5mg/L细胞培养的培养下产生高度纯化、完全加工的KRAS4b。
在开始结构生物学或药物筛选研究之前，仔细的蛋白质表征对于验证重组蛋白的质量至关重要。完全加工的KRAS4b的两个关键参数是验证正确的前阴修饰和可及的法奈酸和卡博甲基化C-总站（FMe）与膜替代品或脂质相互作用。KRAS4b-FMe的电喷雾电离质谱法（ESI-MS）用于测量分子量，并确认存在法内西尔和甲苯甲基修饰。原生质谱法，其中样品喷洒非脱模溶剂，用于证明KRAS4b-FMe也绑定到其国内生产总值的系数。最后，利用表面质子共振光谱测量KRAS4b-FMe与固定脂质体的直接结合。
Protocol
Representative Results
Discussion
如”代表性结果”部分所述，在纯化过程中最关键的步骤是在样品处于低盐时处理。限制样品暴露于小于 200 mM NaCl 的时间将有助于减少降水并提高样品产量。如果配置文件不符合预期，则很难解释 CEX 的结果（参见图 2）。在协议成为常规之前，建议未向前推进的CEX洗脱分数在-80°C处储存，直到完成完整的质量分析，以确保将适当的材料向前推进。除了上述CEX步骤的参数外，该…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢卡里萨·格罗斯、詹·梅尔哈尔科和马特·德鲁在弗雷德里克国家癌症研究实验室的蛋白质表达实验室的克隆和表达支持。该项目已全部或部分由国家癌症研究所、国家卫生研究院的联邦资金供资，合同号为No.HHSN261200800001E。本出版物的内容不一定反映卫生与公众服务部的观点或政策，也不提及商号、商业产品或组织，也不表示美国政府认可
Materials
| 1.8 mL Safe-Lock Tubes, Natural | Eppendorf | 22363204 | |
| 11 mm Cl SS Interlocked Insert Autosampler Vials | Thermo Scientific | 30211SS-1232 | |
| 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) | AVANTI POLAR LIPIDS | 850457 | purchase as liquid stocks in chloroform |
| 1-palmitoyl-2-oleoyl-glycero-3-phospho-L-serine (POPS) | AVANTI POLAR LIPIDS | 840034 | purchase as liquid stocks in chloroform |
| 5427R Centrifuge | Eppendorf | ||
| Acetonitrile, HPLC Grade | Fisher Chemical | A998-1 1L | |
| Ammonium Acetate | Sigma-Aldrich | 09689-250g | |
| Argon gas | Airgas | ARUP | |
| Assay Plate 384 | CORNING | 3544 | |
| Biacore T200 Instrument | GE Healthcare | ||
| Blue Snap-It Seals, T/S | Thermo Scientific | C4011-54B | |
| Branson Ultrasonic Bath | Thermo Fisher | 15-336-1000 | |
| Cation Exchange Chromatography (CEX) column | GE Healthcare Life Sciences | 29018183 | HiPrep SP Sepharose High Performance |
| CHAPS | Sigma | C3023 | |
| Dyna Pro Plate Reader | Wyatt Technologies | ||
| Exactive Plus EMR Mass Spectrometer | Thermo Scientific | ||
| Formic Acid | Sigma-Aldrich | F0507-500Ml | Use Reagent Grade or better |
| Gilson vials 7×14 mm Tubes | GE Healthcare | BR-1002-12 | |
| Glass screw thread vials with PTFE foam liners | Scientific Specialities | B69302 | |
| High speed/benchtop centrifuge | Thermo Fischer Scientific | 05-112-114D | capable of up to 4,000 xg |
| His6-Tobacco Etch Virus (TEV) protease | Addgene | 92414 | Purified as per Raran-Kurussi et al. (2017) Removal of Affinity Tags with TEV Protease. In: Burgess-Brown N. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY |
| Immobilized Metal Affinity Chromatography (IMAC) column | GE Healthcare Life Sciences | 28-9365-51 | HisPrep FF 16/10 |
| In-House Water Supply, Arium Advance | Sartorius Stedim | Resistivity of 18 MΩ0-cm | |
| Lipid extruder set with holder | AVANTI POLAR LIPIDS | 610023 | |
| Liquid nitrogen | Airgas | NI-DEWAR | |
| M110-EH microfluidizer | Microfluidics | ||
| MabPac RP UHPLC Column, 4 um, 3.0 x 50 mm | Thermo Scientific | 088645 | |
| MabPac SEC-1 Column, 5 um, 300 Å, 2.1 x 150 mm | Thermo Scientific | 088790 | |
| MagTran software | Thermo Scientific | ||
| Methanol, HPLC Grade | VWR Chemicals | BDH20864.400 | |
| NGC Chromatography System | BioRad | 78880002 | NGC QuestTM 100 Chromatography system |
| Protease Inhibitor Cocktail without EDTA or other chelators | Millipore Sigma | P8849 | |
| Rubber Caps type 3 | GE Healthcare | BR-1005-02 | |
| Series S Sensor Chip L1 | GE Healthcare | 29104993 | |
| Spectrophotometer | Thermo Fischer Scientific | 13-400-519 | Absorbace at 280nm |
| Ultra-15 Centrifugal Filter Units, 10K NMWL | Millipore Sigma | UFC901008 | PES membrane |
| Ultracel 10K MWCO Ultra 0.5 mL Centrifuge Filters | Amicon | UFC501024 | |
| Ultracentrifuge | Beckman Coulter | Optima – L80K | capable of 100,000 xg |
| Vanquish UHPLC (Pump, Column Hearter, and LC System) | Thermo Scientific | ||
| Vortex Genie 2 | Fisher | 12-812 | |
| Water, HPLC Grade | Sigma-Aldrich | 270733-1L | May use in-house water source (see below) |
| Whatman GD/XP PES 0.45 mm syringe filter | GE Healthcare – Whatman | 6994-2504 | |
| Xcalibur QualBrowser | Thermo Scientific | proteomics software |
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