用于蛋白质组学分析的带标签稳定细胞系的高通量生成。
High-throughput generation of tagged stable cell lines for proteomic analysis.
作者信息
Torres Jorge Z, Miller Julie J, Jackson Peter K
机构信息
Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
出版信息
Proteomics. 2009 May;9(10):2888-91. doi: 10.1002/pmic.200800873.
Abstract
We present an optimized system for rapid generation of localization and affinity purification-tagged mammalian stable cell lines that facilitates complex purification and interacting protein identification. The improved components of this method, including the flexibility of inducible expression, circumvent issues associated with toxicity, clonal selection, sample yields and time to data acquisition. We have applied this method to the study of cell-cycle regulators and novel microtubule-associated proteins.
摘要
我们展示了一种优化系统，用于快速生成带有定位和亲和纯化标签的哺乳动物稳定细胞系，该系统有助于复杂的纯化及相互作用蛋白的鉴定。此方法的改进组件，包括诱导表达的灵活性，规避了与毒性、克隆选择、样品产量及数据获取时间相关的问题。我们已将该方法应用于细胞周期调节因子和新型微管相关蛋白的研究。
相似文献
1
High-throughput generation of tagged stable cell lines for proteomic analysis.用于蛋白质组学分析的带标签稳定细胞系的高通量生成。
Proteomics. 2009 May;9(10):2888-91. doi: 10.1002/pmic.200800873.
2
Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® recombination cloning technology and Flp-In T-REx® lines.利用Gateway®重组克隆技术和Flp-In T-REx®细胞系快速构建用于蛋白质调控表达的稳定哺乳动物细胞系。
Methods Enzymol. 2013;529:99-124. doi: 10.1016/B978-0-12-418687-3.00008-2.
3
Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks.用于研究蛋白质功能、时空定位及蛋白质相互作用网络的可诱导LAP标签稳定细胞系
J Vis Exp. 2016 Dec 24(118):54870. doi: 10.3791/54870.
4
Identification of mammalian protein complexes by lentiviral-based affinity purification and mass spectrometry.通过基于慢病毒的亲和纯化和质谱法鉴定哺乳动物蛋白质复合物。
Methods Mol Biol. 2011;781:31-45. doi: 10.1007/978-1-61779-276-2_2.
5
Methods to Study Translated Pseudogenes: Recombinant Expression and Complementation, Targeted Proteomics, and RNA Profiling.研究翻译假基因的方法：重组表达和互补、靶向蛋白质组学和 RNA 分析。
Methods Mol Biol. 2021;2324:239-254. doi: 10.1007/978-1-0716-1503-4_15.
6
Rapid purification of protein complexes from mammalian cells.从哺乳动物细胞中快速纯化蛋白质复合物。
Nucleic Acids Res. 2000 Jun 15;28(12):E61. doi: 10.1093/nar/28.12.e61.
7
LBT/PTD dual tagged vector for purification, cellular protein delivery and visualization in living cells.用于在活细胞中进行纯化、细胞蛋白质递送和可视化的LBT/PTD双标记载体。
Biochim Biophys Acta. 2007 Feb;1773(2):141-6. doi: 10.1016/j.bbamcr.2006.11.016. Epub 2006 Dec 6.
8
Expression and purification of epitope-tagged multisubunit protein complexes from mammalian cells.从哺乳动物细胞中表达和纯化表位标记的多亚基蛋白复合物
Curr Protoc Mol Biol. 2002 Nov;Chapter 16:Unit 16.13. doi: 10.1002/0471142727.mb1613s60.
9
High-throughput expression, purification, and characterization of recombinant Caenorhabditis elegans proteins.重组秀丽隐杆线虫蛋白质的高通量表达、纯化及特性分析
Biochem Biophys Res Commun. 2003 Aug 8;307(4):928-34. doi: 10.1016/s0006-291x(03)01265-8.
10
Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins.构建一个小型内含肽融合系统，以实现对可溶性蛋白质表达的直接监测和目标蛋白质的快速纯化。
Gene. 2001 Sep 19;275(2):241-52. doi: 10.1016/s0378-1119(01)00663-1.
引用本文的文献
1
Characterization of membrane structures regulating primary ciliogenesis by quantitative isotropic ultrastructure imaging.通过定量各向同性超微结构成像对调节初级纤毛发生的膜结构进行表征。
bioRxiv. 2025 Aug 21:2025.08.20.670930. doi: 10.1101/2025.08.20.670930.
2
The human ciliopathy protein RSG1 links the CPLANE complex to transition zone architecture.人类纤毛病蛋白RSG1将CPLANE复合体与过渡区结构联系起来。
Nat Commun. 2025 Jul 1;16(1):5701. doi: 10.1038/s41467-025-61005-8.
3
Myristoylated Neuronal Calcium Sensor-1 captures the preciliary vesicle at distal appendages.肉豆蔻酰化的神经元钙传感器-1在远端附属物处捕获纤毛前小泡。
Elife. 2025 Jan 30;14:e85998. doi: 10.7554/eLife.85998.
4
DUSP12 promotes cell cycle progression and protects cells from cell death by regulating ZPR9.双特异性磷酸酶12（DUSP12）通过调节ZPR9促进细胞周期进程并保护细胞免于细胞死亡。
bioRxiv. 2025 Jan 14:2025.01.13.632830. doi: 10.1101/2025.01.13.632830.
5
Human REXO4 is Required for Cell Cycle Progression.细胞周期进程需要人类REXO4。
bioRxiv. 2025 Jan 9:2025.01.08.631954. doi: 10.1101/2025.01.08.631954.
6
Cul3 substrate adaptor SPOP targets Nup153 for degradation.Cul3底物衔接蛋白SPOP将核孔蛋白153作为降解靶点。
Mol Biol Cell. 2025 Mar 1;36(3):ar24. doi: 10.1091/mbc.E24-04-0198. Epub 2025 Jan 9.
7
A defined tubby domain β-barrel surface region of TULP3 mediates ciliary trafficking of diverse cargoes.TULP3的一个特定的桶状结构域β-桶表面区域介导多种货物的纤毛运输。
Mol Biol Cell. 2025 Jan 1;36(1):ar1. doi: 10.1091/mbc.E24-09-0426. Epub 2024 Nov 20.
8
Multi-color live-cell fluorescence imaging of primary ciliary membrane assembly and dynamics.多色活细胞荧光成像观察初级纤毛膜的组装和动态变化。
Methods Cell Biol. 2023;176:235-250. doi: 10.1016/bs.mcb.2023.02.001. Epub 2023 Mar 27.
9
Human IFT-A complex structures provide molecular insights into ciliary transport.人IFT-A 复合物结构为纤毛运输提供了分子层面的见解。
Cell Res. 2023 Apr;33(4):288-298. doi: 10.1038/s41422-023-00778-3. Epub 2023 Feb 13.
10
Interactions between TULP3 tubby domain and ARL13B amphipathic helix promote lipidated protein transport to cilia.TULP3 微管域与 ARL13B 两亲性螺旋相互作用促进脂化蛋白向纤毛的转运。
Mol Biol Cell. 2023 Mar 1;34(3):ar18. doi: 10.1091/mbc.E22-10-0473. Epub 2023 Jan 18.
本文引用的文献
1
Katanin regulates dynamics of microtubules and biogenesis of motile cilia.katanin调节微管的动力学和运动性纤毛的生物发生。
J Cell Biol. 2007 Sep 10;178(6):1065-79. doi: 10.1083/jcb.200704021.
2
hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes.人类开放阅读框组v3.1：一个代表超过10000个人类基因的人类开放阅读框资源库。
Genomics. 2007 Mar;89(3):307-15. doi: 10.1016/j.ygeno.2006.11.012. Epub 2007 Jan 5.
3
Katanin controls mitotic and meiotic spindle length.katanin蛋白控制有丝分裂和减数分裂纺锤体的长度。
J Cell Biol. 2006 Dec 18;175(6):881-91. doi: 10.1083/jcb.200608117.
4
Emi1 stably binds and inhibits the anaphase-promoting complex/cyclosome as a pseudosubstrate inhibitor.Emi1作为一种假底物抑制剂稳定结合并抑制后期促进复合物/细胞周期体。
Genes Dev. 2006 Sep 1;20(17):2410-20. doi: 10.1101/gad.1454006. Epub 2006 Aug 18.
5
Microtubules cut and run.微管切断并脱离。
Trends Cell Biol. 2005 Oct;15(10):518-24. doi: 10.1016/j.tcb.2005.08.004.
6
A combined approach for the localization and tandem affinity purification of protein complexes from metazoans.一种用于后生动物蛋白质复合物定位和串联亲和纯化的联合方法。
Sci STKE. 2005 Jan 11;2005(266):pl1. doi: 10.1126/stke.2662005pl1.
7
Human ORFeome version 1.1: a platform for reverse proteomics.人类开放阅读框表达文库1.1版：一个反向蛋白质组学平台。
Genome Res. 2004 Oct;14(10B):2128-35. doi: 10.1101/gr.2973604.
8
ORFeome cloning and systems biology: standardized mass production of the parts from the parts-list.开放阅读框（ORF）文库克隆与系统生物学：依据元件清单进行元件的标准化大规模生产。
Genome Res. 2004 Oct;14(10B):2001-9. doi: 10.1101/gr.2769804.
9
Prophase destruction of Emi1 by the SCF(betaTrCP/Slimb) ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase.SCF（βTrCP/Slimb）泛素连接酶在前期对Emi1的破坏会激活后期促进复合体，以使细胞能够越过前中期继续发展。
Dev Cell. 2003 Jun;4(6):813-26. doi: 10.1016/s1534-5807(03)00153-9.
10
An efficient protein complex purification method for functional proteomics in higher eukaryotes.一种用于高等真核生物功能蛋白质组学的高效蛋白质复合物纯化方法。
Nat Biotechnol. 2003 Jan;21(1):89-92. doi: 10.1038/nbt773. Epub 2002 Dec 16.
Zotero 插件