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Current Protocols in Protein Science最新文献

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Generation of Recombinant Vaccinia Viruses 重组痘苗病毒的产生
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.33
Linda S. Wyatt, Patricia L. Earl, Bernard Moss
{"title":"Generation of Recombinant Vaccinia Viruses","authors":"Linda S. Wyatt,&nbsp;Patricia L. Earl,&nbsp;Bernard Moss","doi":"10.1002/cpps.33","DOIUrl":"10.1002/cpps.33","url":null,"abstract":"<p>This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are described. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.33","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35232423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 65
Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector 杆状病毒感染昆虫幼虫重组人酪氨酸酶的纯化
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.37
Monika B. Dolinska, Paul T. Wingfield, Yuri V. Sergeev
{"title":"Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector","authors":"Monika B. Dolinska,&nbsp;Paul T. Wingfield,&nbsp;Yuri V. Sergeev","doi":"10.1002/cpps.37","DOIUrl":"10.1002/cpps.37","url":null,"abstract":"<p>The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate-limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in <i>Trichoplusia ni</i> larvae, and purified using affinity and size-exclusion chromatography. The purified recombinant human tyrosinase is a soluble monomeric glycoprotein with an activity that mirrors the tyrosinase in vivo function. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.37","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35232424","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 12
Screening and Identifying Membrane Proteins Favorable for Crystallization 有利于结晶的膜蛋白的筛选与鉴定
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.40
Jared Kim, Ho Leung Ng
{"title":"Screening and Identifying Membrane Proteins Favorable for Crystallization","authors":"Jared Kim,&nbsp;Ho Leung Ng","doi":"10.1002/cpps.40","DOIUrl":"10.1002/cpps.40","url":null,"abstract":"<p>This unit addresses several critical challenges associated with membrane protein crystallography by screening membrane proteins from <i>Escherichia coli</i>, <i>Saccharomyces cerevisiae</i>, and <i>Sus scrofa</i> cerebral tissue for biochemical properties favorable for crystallization. First, a tissue sample or cell pellet is obtained. The cells are isolated, washed, and then lysed either by sonication, bead beating, or manual homogenization. Membrane proteins are fractionated from the lysates by centrifugation and solubilized in a mild detergent suitable for crystallization, such as <i>n</i>-dodecyl-β-maltoside (DDM). Detergent extracts are then centrifuged, heat precipitated, and filtered to remove insoluble, thermally unstable, and/or aggregated proteins. Samples are then prepared for analysis by mass spectrometry: proteins are precipitated by methanol/chloroform extraction and subjected to reduction, alkylation, and protease digestion. The resulting peptides are passed through a detergent removal column, desalted, rehydrated in 0.1% formic acid (v/v), and identified by LC-MS/MS. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"90 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.40","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35511934","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Preparation of Cell Cultures and Vaccinia Virus Stocks 细胞培养和牛痘病毒库的制备
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.34
Catherine A. Cotter, Patricia L. Earl, Linda S. Wyatt, Bernard Moss
{"title":"Preparation of Cell Cultures and Vaccinia Virus Stocks","authors":"Catherine A. Cotter,&nbsp;Patricia L. Earl,&nbsp;Linda S. Wyatt,&nbsp;Bernard Moss","doi":"10.1002/cpps.34","DOIUrl":"10.1002/cpps.34","url":null,"abstract":"<p>The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.34","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35371492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 24
Expression and Purification of Protein Complexes Suitable for Structural Studies Using Mammalian HEK 293F Cells 适用于哺乳动物HEK 293F细胞结构研究的蛋白复合物的表达和纯化
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.44
Irene Nigi, Louise Fairall, John W.R. Schwabe
{"title":"Expression and Purification of Protein Complexes Suitable for Structural Studies Using Mammalian HEK 293F Cells","authors":"Irene Nigi,&nbsp;Louise Fairall,&nbsp;John W.R. Schwabe","doi":"10.1002/cpps.44","DOIUrl":"10.1002/cpps.44","url":null,"abstract":"<p>Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not produce significant yields of soluble protein. This may result from failure to efficiently transcribe/translate the required protein or may result from the formation of insoluble aggregates known as inclusion bodies. Mammalian expression systems can often produce natively folded proteins, sometimes with native post-translational modifications. However, such expression systems are underutilized due to the perception that they are costly, technically challenging and result in limited protein yields. In fact, HEK 293F cells are straightforward to grow, transfect with high efficiency and often produce significant yields of recombinant proteins. In this unit, we describe a method to express and purify milligram quantities of a human protein complex from HEK 293F cells grown in suspension transiently transfected with the appropriate plasmids. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"90 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.44","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35511935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 10
Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag 利用肝素结合亲和标签简单高效地纯化重组蛋白
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.41
Srinivas Jayanthi, Ravi Kumar Gundampati, Thallapuranam Krishnaswamy Suresh Kumar
{"title":"Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag","authors":"Srinivas Jayanthi,&nbsp;Ravi Kumar Gundampati,&nbsp;Thallapuranam Krishnaswamy Suresh Kumar","doi":"10.1002/cpps.41","DOIUrl":"10.1002/cpps.41","url":null,"abstract":"<p>Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"90 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.41","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35212096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 6
Visualization of Protein Interactions in Living Cells Using Bimolecular Luminescence Complementation (BiLC) 利用双分子发光互补(BiLC)可视化活细胞中蛋白质相互作用
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.42
Lisette G.G.C. Verhoef, Mark Wade
{"title":"Visualization of Protein Interactions in Living Cells Using Bimolecular Luminescence Complementation (BiLC)","authors":"Lisette G.G.C. Verhoef,&nbsp;Mark Wade","doi":"10.1002/cpps.42","DOIUrl":"10.1002/cpps.42","url":null,"abstract":"<p>The number of intracellular protein-protein interactions (PPIs) far exceeds the total number of proteins encoded by the genome. Dynamic cellular PPI networks respond to external stimuli and endogenous metabolism in order to maintain homeostasis. Many PPIs are directly involved in disease pathogenesis and/or resistance to therapeutics; they therefore represent potential drug targets. A technology generally termed ‘bimolecular complementation’ relies on the physical splitting of a molecular reporter (such as a fluorescent or luminescent protein) and fusion of the resulting two fragments to a pair of interacting proteins. When these proteins interact, they effectively reconstitute the activity of the molecular reporter (typically leading to increased fluorescence or luminescence). This unit describes the selection and development of bimolecular luminescence complementation (BiLC) assays for reporting intracellular PPIs, and provides examples in which BiLC was used to identify small molecules that can modulate PPIs. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"90 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.42","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35212095","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Protein-Lipid Interaction by Fluorescence (PLIF) to Characterize and Screen for Inhibitors of Protein-Phosphoinositide Interactions 蛋白-脂质相互作用荧光(PLIF)表征和筛选蛋白-磷酸肌苷相互作用抑制剂
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.35
Laurie Ceccato, Mélanie Mansat, Bernard Payrastre, Frédérique Gaits-Iacovoni, Julien Viaud
{"title":"Protein-Lipid Interaction by Fluorescence (PLIF) to Characterize and Screen for Inhibitors of Protein-Phosphoinositide Interactions","authors":"Laurie Ceccato,&nbsp;Mélanie Mansat,&nbsp;Bernard Payrastre,&nbsp;Frédérique Gaits-Iacovoni,&nbsp;Julien Viaud","doi":"10.1002/cpps.35","DOIUrl":"10.1002/cpps.35","url":null,"abstract":"<p>Phosphoinositides are key signaling and regulatory phospholipids that mediate important pathophysiological processes. This is achieved through the interaction of their phosphorylated inositol head group with a wide range of protein domains. Therefore, being able to determine the phosphoinositide specificity for effector protein is essential to the understanding of its cellular function. This unit describes a novel method named Protein-Lipid Interaction by Fluorescence, or PLIF. PLIF is a fast, reliable and high throughput assay that allows determination of the phosphoinositide specificity of proteins, simultaneously providing relative affinities. In addition, PLIF is suitable for screening inhibitors of protein- phosphoinositide interaction, allowing identification of potential pharmacological compounds. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.35","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35371491","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Analysis of Disulfide Bond Formation 二硫键形成的分析
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.43
Ineke Braakman, Lydia Lamriben, Guus van Zadelhoff, Daniel N. Hebert
{"title":"Analysis of Disulfide Bond Formation","authors":"Ineke Braakman,&nbsp;Lydia Lamriben,&nbsp;Guus van Zadelhoff,&nbsp;Daniel N. Hebert","doi":"10.1002/cpps.43","DOIUrl":"10.1002/cpps.43","url":null,"abstract":"<p>In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS-PAGE with and without prior reduction. The difference in mobility observed between the gels with nonreduced and reduced samples is due to disulfide bonds in the nonreduced protein. An additional support protocol is included that uses PEG-maleimide to modify free thiols and follow disulfide-bond formation by SDS-PAGE. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"90 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.43","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35212093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 21
Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System 利用Expresso®溶解度和表达筛选系统筛选重组蛋白在大肠杆菌中的表达
Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.39
Eric J. Steinmetz, Michele E. Auldridge
{"title":"Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System","authors":"Eric J. Steinmetz,&nbsp;Michele E. Auldridge","doi":"10.1002/cpps.39","DOIUrl":"10.1002/cpps.39","url":null,"abstract":"<p>The simplicity, speed, and low cost of bacterial culture make <i>E. coli</i> the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"90 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.39","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35212094","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 8
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