{"title":"利用大肠杆菌表达系统表达和纯化人 U1-70K (snRNP70) 及其 BAD 结构域。","authors":"Trenton M. Paul, Shariq Jamal, Ethan Ekpenyong, Peter Prevelige, Talia E. Fargason, Zihan Zhang, Jun Zhang","doi":"10.1002/cpz1.1059","DOIUrl":null,"url":null,"abstract":"<p>U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Expression and purification of full-length U1-70K from <i>E. coli</i></p><p><b>Support Protocol 1</b>: Making chemically competent BL21 Star pRARE/pBB535 cells</p><p><b>Basic Protocol 2</b>: Phosphorylation of full-length U1-70K using SRPK1</p><p><b>Support Protocol 2</b>: Purification of SRPK1</p><p><b>Basic Protocol 3</b>: Expression and purification of U1-70K BAD1 from <i>E. coli</i></p><p><b>Basic Protocol 4</b>: Phosphorylation of U1-70K BAD1 using SRPK1</p><p><b>Basic Protocol 5</b>: Expression and purification of U1-70K BAD2 from <i>E. coli</i></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.1059","citationCount":"0","resultStr":"{\"title\":\"Expression and Purification of Human U1-70K (snRNP70) and its BAD Domains Using an E. coli Expression System\",\"authors\":\"Trenton M. 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In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Expression and purification of full-length U1-70K from <i>E. coli</i></p><p><b>Support Protocol 1</b>: Making chemically competent BL21 Star pRARE/pBB535 cells</p><p><b>Basic Protocol 2</b>: Phosphorylation of full-length U1-70K using SRPK1</p><p><b>Support Protocol 2</b>: Purification of SRPK1</p><p><b>Basic Protocol 3</b>: Expression and purification of U1-70K BAD1 from <i>E. coli</i></p><p><b>Basic Protocol 4</b>: Phosphorylation of U1-70K BAD1 using SRPK1</p><p><b>Basic Protocol 5</b>: Expression and purification of U1-70K BAD2 from <i>E. coli</i></p>\",\"PeriodicalId\":93970,\"journal\":{\"name\":\"Current protocols\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-06-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.1059\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current protocols\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.1059\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.1059","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Expression and Purification of Human U1-70K (snRNP70) and its BAD Domains Using an E. coli Expression System
U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.
Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli
Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells
Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1
Support Protocol 2: Purification of SRPK1
Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli
Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1
Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli