Name: fully processed recombinant kras4b: isolating and characterizing the farnesylated and methylated protein
Uploaded: 2020-01-16T14:00:00
Description: Watch this Scientific Journal Video about Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein at JoVE.com

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

1. 蛋白质纯化
<ol><li>准备缓冲区 A_H，如表1所示。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>缓冲液</td>
			<td>缓冲剂（所有 20 mM）</td>
			<td>
pH
</td>
			<td>纳克莱 （mM）</td>
			<td>伊米达索 （mM）</td>
			<td>MgCl2</td>
			<td>
TCEP</...

<ol>
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A., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+prenylation+and+synaptic+plasticity:+implications+for+Alzheimer's+disease.">Protein prenylation and synaptic plasticity: implications for Alzheimer's disease.</a> Molecular Neurobiology. 50 (1), 177-185 (2014).</li><li>Hottman, D. A., Chernick, D., Cheng, S., Wang, Z., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL+and+cognition+in+neurodegenerative+disorders.">HDL and cognition in neurodegenerative disorders.</a> Neurobiology of Disease. 72, 22-36 (2014).</li><li>Nakagami, H., Jensen, K. S., Liao, J. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+protein+prenylation+for+cancer+therapy.">Targeting protein prenylation for cancer therapy.</a> Nature Reviews Cancer. 11 (11), 775-791 (2011).</li><li>Kho, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tagging-via-substrate+technology+for+detection+and+proteomics+of+farnesylated+proteins.">A tagging-via-substrate technology for detection and proteomics of farnesylated proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (34), 12479-12484 (2004).</li><li>Armstrong, S. A., Hannah, V. C., Goldstein, J. L., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAAX+geranylgeranyl+transferase+transfers+farnesyl+as+efficiently+as+geranylgeranyl+to+RhoB.">CAAX geranylgeranyl transferase transfers farnesyl as efficiently as geranylgeranyl to RhoB.</a> Journal of Biological Chemistry. 270 (14), 7864-7868 (1995).</li><li>Stephen, A. G., Esposito, D., Bagni, R. K., McCormick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dragging+ras+back+in+the+ring.">Dragging ras back in the ring.</a> Cancer Cell. 25 (3), 272-281 (2014).</li><li>Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+in+Membrane+Biochemistry+and+Biophysics.">Nanodiscs in Membrane Biochemistry and Biophysics.</a> Chemical Reviews. 117 (6), 4669-4713 (2017).</li><li>Bao, H., Duong, F., Chan, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Step-by-step+Method+for+the+Reconstitution+of+an+ABC+Transporter+into+Nanodisc+Lipid+Particles.">A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles.</a> Journal of Visualized Experiments. (66), e3910 (2012).</li><li>Dementiev, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K-Ras4B+lipoprotein+synthesis:+biochemical+characterization,+functional+properties,+and+dimer+formation.">K-Ras4B lipoprotein synthesis: biochemical characterization, functional properties, and dimer formation.</a> Protein Expression and Purification. 84 (1), 86-93 (2012).</li><li>Lowe, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+recombinant+human+Kirsten-ras+(4B)+p21+produced+at+high+levels+in+Escherichia+coli+and+insect+baculovirus+expression+systems.">Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems.</a> Journal of Biological Chemistry. 266 (3), 1672-1678 (1991).</li><li>Gillette, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Farnesylated+and+Methylated+Proteins+in+an+Engineered+Insect+Cell+System.">Production of Farnesylated and Methylated Proteins in an Engineered Insect Cell System.</a> Methods in Molecular Biology. 2009, 259-277 (2019).</li><li>Agamasu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KRAS+Prenylation+Is+Required+for+Bivalent+Binding+with+Calmodulin+in+a+Nucleotide-Independent+Manner.">KRAS Prenylation Is Required for Bivalent Binding with Calmodulin in a Nucleotide-Independent Manner.</a> Biophysical Journal. 116 (6), 1049-1063 (2019).</li><li>Talsania, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Assembly+and+Annotation+of+the+Trichoplusia+ni+Tni-FNL+Insect+Cell+Line+Enabled+by+Long-Read+Technologies.">Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies.</a> Genes. 10 (2), 79 (2019).</li><li>Spencer-Smith, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+RAS+function+through+targeting+an+allosteric+regulatory+site.">Inhibition of RAS function through targeting an allosteric regulatory site.</a> Nature Chemical Biology. 13 (1), 62-68 (2016).</li><li>Lowe, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+polyisoprenylated+Ras+proteins+in+the+insect/baculovirus+system.">Expression of polyisoprenylated Ras proteins in the insect/baculovirus system.</a> Biochemical Society Transactions. 20 (2), 484-487 (1992).</li><li>Dharmaiaha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+recognition+of+farnesylated+and+methylated+KRAS4b+by+PDE&#948;.">Structural basis of recognition of farnesylated and methylated KRAS4b by PDE&#948;.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (44), 6766-6775 (2016).</li><li>Abdiche, Y. N., Myszka, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+mechanism+of+drug/Lipid+membrane+interactions+using+Biacore.">Probing the mechanism of drug/Lipid membrane interactions using Biacore.</a> Analytical Biochemistry. 328 (2), 233-243 (2004).</li><li>Fisher, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+interactions+of+HIV-1+nucleocapsid+protein+with+oligonucleotides.">Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides.</a> Nucleic Acids Research. 34 (2), 472-484 (2006).</li><li>Lakshman, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+biophysical+analysis+defines+key+components+modulating+recruitment+of+the+GTPase+KRAS+to+the+plasma+membrane.">Quantitative biophysical analysis defines key components modulating recruitment of the GTPase KRAS to the plasma membrane.</a> Journal of Biological Chemistry. 294, 2193-2207 (2019).</li></ol>

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.8 mL Safe-Lock Tubes, Natural</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>11 mm Cl SS Interlocked Insert Autosampler Vials</td>
			<td>Thermo Scientific</td>
			<td>30211SS-1232</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td>
			<td>AVANTI POLAR LIPIDS</td>
			<td>850457</td>
			<td>purchase as liquid stocks in chloroform</td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phospho-L-serine (POPS)</td>
			<td>AVANTI POLAR LIPIDS</td>
			<td>840034</td>
			<td>purchase as liquid stocks in chloroform</td>
		</tr>
		<tr>
			<td>5427R Centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, HPLC Grade</td>
			<td>Fisher Chemical</td>
			<td>A998-1 1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>09689-250g</td>
			<td></td>
		</tr>
		<tr>
			<td>Argon gas</td>
			<td>Airgas</td>
			<td>ARUP</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Plate 384</td>
			<td>CORNING</td>
			<td>3544</td>
			<td></td>
		</tr>
		<tr>
			<td>Biacore T200 Instrument</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Snap-It Seals, T/S</td>
			<td>Thermo Scientific</td>
			<td>C4011-54B</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson Ultrasonic Bath</td>
			<td>Thermo Fisher</td>
			<td>15-336-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cation Exchange Chromatography (CEX) column</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29018183</td>
			<td>HiPrep SP Sepharose High Performance</td>
		</tr>
		<tr>
			<td>CHAPS</td>
			<td>Sigma</td>
			<td>C3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Dyna Pro Plate Reader</td>
			<td>Wyatt Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Exactive Plus EMR Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>F0507-500Ml</td>
			<td>Use Reagent Grade or better</td>
		</tr>
		<tr>
			<td>Gilson vials 7x14 mm Tubes</td>
			<td>GE Healthcare</td>
			<td>BR-1002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass screw thread vials with PTFE foam liners</td>
			<td>Scientific Specialities</td>
			<td>B69302</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed/benchtop centrifuge</td>
			<td>Thermo Fischer Scientific</td>
			<td>05-112-114D</td>
			<td>capable of up to 4,000 xg</td>
		</tr>
		<tr>
			<td>His6-Tobacco Etch Virus (TEV) protease</td>
			<td>Addgene</td>
			<td>92414</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Immobilized Metal Affinity Chromatography (IMAC) column</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9365-51</td>
			<td>HisPrep FF 16/10</td>
		</tr>
		<tr>
			<td>In-House Water Supply, Arium Advance</td>
			<td>Sartorius Stedim</td>
			<td></td>
			<td>Resistivity of 18 M&#937;0-cm</td>
		</tr>
		<tr>
			<td>Lipid extruder set with holder</td>
			<td>AVANTI POLAR LIPIDS</td>
			<td>610023</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Airgas</td>
			<td>NI-DEWAR</td>
			<td></td>
		</tr>
		<tr>
			<td>M110-EH microfluidizer</td>
			<td>Microfluidics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MabPac RP UHPLC Column, 4 um, 3.0 x 50 mm</td>
			<td>Thermo Scientific</td>
			<td>088645</td>
			<td></td>
		</tr>
		<tr>
			<td>MabPac SEC-1 Column, 5 um, 300 &#197;, 2.1 x 150 mm</td>
			<td>Thermo Scientific</td>
			<td>088790</td>
			<td></td>
		</tr>
		<tr>
			<td>MagTran software</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, HPLC Grade</td>
			<td>VWR Chemicals</td>
			<td>BDH20864.400</td>
			<td></td>
		</tr>
		<tr>
			<td>NGC Chromatography System</td>
			<td>BioRad</td>
			<td>78880002</td>
			<td>NGC QuestTM 100 Chromatography system</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail without EDTA or other chelators</td>
			<td>Millipore Sigma</td>
			<td>P8849</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber Caps type 3</td>
			<td>GE Healthcare</td>
			<td>BR-1005-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Series S Sensor Chip L1</td>
			<td>GE Healthcare</td>
			<td>29104993</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>13-400-519</td>
			<td>Absorbace at 280nm</td>
		</tr>
		<tr>
			<td>Ultra-15 Centrifugal Filter Units, 10K NMWL</td>
			<td>Millipore Sigma</td>
			<td>UFC901008</td>
			<td>PES membrane</td>
		</tr>
		<tr>
			<td>Ultracel 10K MWCO Ultra 0.5 mL Centrifuge Filters</td>
			<td>Amicon</td>
			<td>UFC501024</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima - L80K</td>
			<td>capable of 100,000 xg</td>
		</tr>
		<tr>
			<td>Vanquish UHPLC (Pump, Column Hearter, and LC System)</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Fisher</td>
			<td>12-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, HPLC Grade</td>
			<td>Sigma-Aldrich</td>
			<td>270733-1L</td>
			<td>May use in-house water source (see below)</td>
		</tr>
		<tr>
			<td>Whatman GD/XP PES 0.45 mm syringe filter</td>
			<td>GE Healthcare - Whatman</td>
			<td>6994-2504</td>
			<td></td>
		</tr>
		<tr>
			<td>Xcalibur QualBrowser</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>proteomics software</td>
		</tr>
	</tbody>
</table>

fully processed recombinant kras4b: isolating and characterizing the farnesylated and methylated protein

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。

该协议中最大的变量之一是表达靶蛋白（His6-MBP-tev-KRAS4b）的量。该协议是利用从Trichoplusia ni细胞系Tni-FNL17分离物开发的，该基体适应悬浮生长，并从血清中分离。鉴于使用巴库病毒表达系统在各种昆虫细胞系报告的各种结果范围广泛，建议使用Tni-FNL，至少在最初用于生产KRAS4b-FMe。
迁移到 +65 kDa 的深色蛋白质在澄清的解液和洗脱分数中应明显（<strong ...

Watch this Scientific Journal Video about Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein at JoVE.com

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

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

完全加工重组KRAS4b：分离和描述脱脂和甲基化蛋白

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

我们的协议允许在高产量下分离和分离正确处理的KRAS。正宗法化和碳化 KRAS 的生产使得可以像在哺乳动物细胞中一样进行膜中包含 KRAS 的实验。该协议的高产量使它与以前的工作不同。我们通常每升表达材料纯化三到五毫克蛋白质。这使我们能够进行各种生物物理和结构生物学实验。KRAS在20%的癌症和90%的胰腺癌中发生变异。因此，有一个协议，以产生真正的加工蛋白质是相当有用的开发药物屏幕，以便你可以研究实际蛋白质，因为它将在癌细胞的膜上。该协议对于研究其他在细胞中被法化和完全加工的蛋白质也非常有用。因此，所有你需要做的是交换出KRAS与感兴趣的蛋白质，并把它放在杆菌病毒。关键步骤是围绕阳离子交换色谱的。使用适当的列并限制蛋白质在低盐缓冲液中的时间是成功的关键。了解降水不是雪球类型的事件有助于指导那些新到协议。这通过显示需要将阳十一交换柱负载划分为多个较小的负载而得到加强。在细胞分解后，澄清乳酸，通过IMAC纯化目标蛋白质，使用SDS页面和库马西染色分析蛋白质分数。将峰值分数池池，在四摄氏度下将池对两升缓冲 D 进行一夜。第二天，从透析中取出样品，以4000次g离心10分钟，取出任何沉淀物。最终的拨号和澄清样本通常仍然模糊不清，但可以应用于 CEX 列，而无需进一步处理。用三列缓冲液G洗涤20毫升阳离子交换柱，然后用三列缓冲液F.Next，通过加入20毫升缓冲器E，将透析样品的20毫升稀释到100毫升的氯化钠浓度，最后为100毫升的氯化钠浓度，然后将样品应用到交换柱上。稀释少量样品而不是一次全部稀释至关重要，稀释后的样品应立即应用于柱，以限制蛋白质的沉淀。继续用刚稀释的样品加载柱，因为之前的稀释接近负载的末尾。然后用缓冲 F 将柱洗到基线吸收度 280，这通常需要三个列卷。从柱中用400毫升梯度从缓冲液F到65%缓冲G的蛋白质，以6毫升分数收集埃卢因。梯度完成后，继续清洗列，以增加 1.5 列卷 65%缓冲 G. 选择基于 SDS 页面和 Coomassie Blue 污渍分析的正分数，以及检查色谱图的 UV 轨迹。然后根据手稿指示将蛋白质汇集并与 His6 TEV 蛋白酶一起消化。消化和透析后，将蛋白质加载到20毫升的IMAC柱上，以每分钟3毫升的速度与缓冲液 A进行平衡。在此色谱中收集七毫升分数，用三列缓冲器 A 或直到达到基线吸收度之前清洗柱。用缓冲液C的五列体积梯度从0%至10%将目标蛋白从0%至10%，并收集7毫升分数。然后使用 SDS 页确定正分数。当使用SDS页面分析时，澄清的芦酸应包含一个暗带，该暗带会迁移到约65千吨，与融合蛋白 His6-MBP-tev-KRAS4b 相对应。共同表达的FNTAb迁移到48千吨。纯化中的 CEX 步骤至关重要，因为它可降低蛋白解的程度并丰富完全加工的蛋白质，但却是协议中最复杂的部分。此步骤的典型结果具有 KRAS4b-FMe 的突出峰值三，但洗脱配置文件可以是可变的。使用ESIMS进行的完整性质量分析证实了蛋白质的精确分子质量，从而确认了远尼化或碳甲基化的相对比例。虽然典型的最终地段包含一些可检测的KRAS4b-FARN，但根据此分析，这一比例在峰值高度方面低于15%。原生质量分析用于确定与 GDP 绑定的 KRAS4b 的质量。将样品溶剂交换为醋酸铵，以确保更柔和的电离，使原生复合物保持完好无损。用表面质膜共振测量了KRAS4b-FMe和脂质体之间的相互作用，以验证KRAS4b-FMe与膜结合所需的法尼化和碳氧甲基化。正如所料，KRAS4b-FMe 绑定到脂质体，而未处理的 KRAS4b 没有。尽量减少蛋白质暴露于低盐的时间是协议中影响产量的单一最重要的方面。在缓冲液E稀释后，该蛋白的盐浓度仅处于这种低盐浓度，该蛋白可用于各种生物物理实验，这些实验使用脂质体或纳米光片等膜仿生学来研究KRAS与哪些脂质相互作用。利用这些数据，我们可以开始推断KRAS如何与细胞的等离子膜相互作用。使用纯化的KRAS-FMe，我们的同事能够解决KRAS的X射线晶体结构在复杂的陪护，这是特定于这种蛋白质的法尼化和甲基化形式。

Research

JoVE Journal

Biochemistry

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

制备及脂质立方相反应蛋白微晶交付串行飞秒晶体

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

科研

生物化学

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

检查蛋白酶体大会与重组古菌蛋白酶体和非变性PAGE：该案例相结合的办法

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

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

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

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 ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

生物大分子晶体的完全自主表征及数据采集

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

离子交换色谱 (IEX) 与多角度光散射 (ALS) 相结合, 用于蛋白质分离和表征

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

通过红外纳米光谱和原子力显微镜对单个蛋白质聚合进行特征化

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...