Current Protocols in Protein Science最新文献_第2页

Assessment of the Higher-Order Structure of Formulated Monoclonal Antibody Therapeutics by 2D Methyl Correlated NMR and Principal Component Analysis 用二维甲基相关核磁共振和主成分分析评价配方单克隆抗体治疗剂的高阶结构

Current Protocols in Protein Science Pub Date : 2020-05-14 DOI: 10.1002/cpps.105

Luke W. Arbogast, Frank Delaglio, Robert G. Brinson, John P. Marino

{"title":"Assessment of the Higher-Order Structure of Formulated Monoclonal Antibody Therapeutics by 2D Methyl Correlated NMR and Principal Component Analysis","authors":"Luke W. Arbogast, Frank Delaglio, Robert G. Brinson, John P. Marino","doi":"10.1002/cpps.105","DOIUrl":"10.1002/cpps.105","url":null,"abstract":"Characterization of the higher-order structure (HOS) of protein therapeutics, and in particular of monoclonal antibodies, by 2D 1H-13C methyl correlated NMR has been demonstrated as precise and robust. Such characterization can be greatly enhanced when collections of spectra are analyzed using multivariate approaches such as principal component analysis (PCA), allowing for the detection and identification of small structural differences in drug substance that may otherwise fall below the limit of detection of conventional spectral analysis. A major limitation to this approach is the presence of aliphatic signals from formulation or excipient components, which result in spectral interference with the protein signal of interest; however, the recently described Selective Excipient Reduction and Removal (SIERRA) filter greatly reduces this issue. Here we will outline how basic 2D 1H-13C methyl−correlated NMR may be combined with the SIERRA approach to collect ‘clean’ NMR spectra of formulated monoclonal antibody therapeutics (i.e., drug substance spectra free of interfering component signals), and how series of such spectra may be used for HOS characterization by direct PCA of the series spectral matrix. © 2020 U.S. Government.Basic Protocol 1: NMR data acquisitionBasic Protocol 2: Full spectral matrix data processing and analysisSupport Protocol: Data visualization and cluster analysis","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"100 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.105","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37935530","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

Two-Step Preparation of Highly Pure, Soluble HIV Protease from Inclusion Bodies Recombinantly Expressed in Escherichia coli 从大肠杆菌中重组表达的包涵体两步制备高纯度、可溶性HIV蛋白酶

Current Protocols in Protein Science Pub Date : 2020-04-27 DOI: 10.1002/cpps.106

Dean Sherry, Roland Worth, Yasien Sayed

{"title":"Two-Step Preparation of Highly Pure, Soluble HIV Protease from Inclusion Bodies Recombinantly Expressed in Escherichia coli","authors":"Dean Sherry, Roland Worth, Yasien Sayed","doi":"10.1002/cpps.106","DOIUrl":"10.1002/cpps.106","url":null,"abstract":"Heterologous expression of exogenous proteases in Escherichia coli often results in the formation of insoluble inclusion bodies. When sequestered into inclusion bodies, the functionality of the proteases is minimized. To be characterized structurally and functionally, however, proteases must be obtained in their native conformation. HIV protease is readily expressed as inclusion bodies, but must be recovered from the inclusion bodies. This protocol describes an efficient method for recovering HIV protease from inclusion bodies, as well as refolding and purifying the protein. HIV protease–containing inclusion bodies are treated with 8 M urea and purified via cation-exchange chromatography. Subsequent refolding by buffer exchange via dialysis and further purification by anion-exchange chromatography produces highly pure HIV protease that is functionally active. © 2020 by John Wiley & Sons, Inc.Basic Protocol: Recovery, refolding, and purification of HIV protease from inclusion bodiesSupport Protocol 1: Expression and extraction of inclusion bodies containing HIV protease expressed in Escherichia coliSupport Protocol 2: Determination of the active site concentration of HIV protease via isothermal titration calorimetry","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"100 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37876265","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

Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations 毕赤酵母重组膜蛋白的制备及其分子研究

Current Protocols in Protein Science Pub Date : 2020-04-14 DOI: 10.1002/cpps.104

Lucile Guyot, Lucie Hartmann, Sarah Mohammed-Bouteben, Lydia Caro, Renaud Wagner

{"title":"Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations","authors":"Lucile Guyot, Lucie Hartmann, Sarah Mohammed-Bouteben, Lydia Caro, Renaud Wagner","doi":"10.1002/cpps.104","DOIUrl":"10.1002/cpps.104","url":null,"abstract":"Pichia pastoris is a eukaryotic microorganism reputed for its ability to mass-produce recombinant proteins, including integral membrane proteins, for various applications. This article details a series of protocols that progress towards the production of integral membrane proteins, their extraction and purification in the presence of detergents, and their eventual reconstitution in lipid nanoparticles. These basic procedures can be further optimized to provide integral membrane protein samples that are compatible with a number of structural and/or functional investigations at the molecular level. Each protocol provides general guidelines, technical hints, and specific recommendations, and is illustrated with case studies corresponding to several representative mammalian proteins. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Production of membrane proteins in a P. pastoris recombinant clone using methanol inductionBasic Protocol 2: Preparation of whole-membrane fractionsAlternate Protocol 1: Preparation of yeast protoplastsBasic Protocol 3: Extraction of membrane proteins from whole-membrane fractionsBasic Protocol 4: Purification of membrane proteinsAlternate Protocol 2: Purification of membrane proteins from yeast protoplastsAlternate Protocol 3: Simultaneous protoplast preparation and membrane solubilization for purification of membrane proteinsBasic Protocol 5: Reconstitution of detergent-purified membrane proteins in lipid nanoparticles","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"100 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37833903","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: 4

GlycoBIST: A System for Automatic Glycan Profiling of a Target Protein Using Milli-Bead Array in a Tip GlycoBIST:一种在尖端使用毫微珠阵列对目标蛋白进行自动糖谱分析的系统

Current Protocols in Protein Science Pub Date : 2020-02-19 DOI: 10.1002/cpps.103

Hiroko Shimazaki, Ayaka Ono, Masako Tsuruga, Aya Ueki, Shiori Koseki-Kuno, Takako Toyoda, Kozue Saito, Kazumi Sawakami, Minoru Kariya, Osamu Segawa, Kazuhiro Nakamura, Michinori Koizuka, Atsushi Kuno

{"title":"GlycoBIST: A System for Automatic Glycan Profiling of a Target Protein Using Milli-Bead Array in a Tip","authors":"Hiroko Shimazaki, Ayaka Ono, Masako Tsuruga, Aya Ueki, Shiori Koseki-Kuno, Takako Toyoda, Kozue Saito, Kazumi Sawakami, Minoru Kariya, Osamu Segawa, Kazuhiro Nakamura, Michinori Koizuka, Atsushi Kuno","doi":"10.1002/cpps.103","DOIUrl":"10.1002/cpps.103","url":null,"abstract":"Lectin is a biomolecule that recognizes a specific part of glycans and, thus, has been used widely as a probe for glycoprotein analysis. Owing to the wide repertoire in nature combined with the recent two decades of advances in microarray technology, the multiplexed use of lectins has been widely used for glycan profiling of endogenous proteins. Because protein glycosylation is recognized as being biologically important and is expected to be a reliable disease marker, lectin microarray analysis with highly sensitive detection has been used to discover disease-relevant glycosylation alterations. However, the conventional system is limited to research purposes; thus, its implementation in clinical settings is warranted. Here, we provide an automatic glycan profiling method using GlycoBIST. A unique array format is used for 10-plexed lectin–glycoprotein interaction analysis on 1-mm-sized beads, which are arranged vertically in a capillary-shaped plastic tip. Using a one-boxed autopipetting machine, the whole process (including interaction, washing, and detection) is performed automatically and serially, resulting in reproducible measurements. In this article, a typical method for glycan profiling of a purified glycoprotein and the fabrication of GlycoBIST tips is explained. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Fabrication of a GlycoBIST tipBasic Protocol 2: Automatic profiling of a target glycoprotein using GlycoBIST","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"99 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.103","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37657542","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: 2

Magnetic Resonance Relaxometry for Determination of Protein Concentration and Aggregation 磁共振松弛法测定蛋白质浓度和聚集

Current Protocols in Protein Science Pub Date : 2019-12-23 DOI: 10.1002/cpps.102

Marc B. Taraban, Katharine T. Briggs, Yihua Bruce Yu

{"title":"Magnetic Resonance Relaxometry for Determination of Protein Concentration and Aggregation","authors":"Marc B. Taraban, Katharine T. Briggs, Yihua Bruce Yu","doi":"10.1002/cpps.102","DOIUrl":"10.1002/cpps.102","url":null,"abstract":"The water-proton signal, overwhelmingly considered a nuisance in nuclear magnetic resonance spectroscopy, is advantageously used as a tool to assess protein concentration and to detect protein aggregates in aqueous solutions. The protocols in this article describe use of the water-proton transverse relaxation rate to determine concentration and aggregate content in protein solutions. Detailed recommendations and description of the parameter settings and data processing ensure successful implementation of this technique, even by a user with limited experience in magnetic resonance relaxometry. All measurements are done noninvasively, in a sealed container, without sampling or otherwise aliquoting the solution. The magnetic resonance relaxometry approach offered in this article could be advantageous for analysis of biologics formulations or when use of conventional analytical techniques is not possible. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: Nuclear magnetic resonance (NMR) relaxometry to measure protein concentrationBasic Protocol 2: NMR relaxometry to measure protein aggregation","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"99 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.102","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37485011","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

Isolation of High-Quality RNA from Pichia pastoris 毕赤酵母高质量RNA的分离

Current Protocols in Protein Science Pub Date : 2019-12-09 DOI: 10.1002/cpps.101

Sibel Öztürk, İrem Demir, Pınar Çalık

{"title":"Isolation of High-Quality RNA from Pichia pastoris","authors":"Sibel Öztürk, İrem Demir, Pınar Çalık","doi":"10.1002/cpps.101","DOIUrl":"10.1002/cpps.101","url":null,"abstract":"Analysis of RNA structuromes provides new insights into cellular processes, enabling systems biology and biotechnology researchers to calculate promoter and terminator strengths and to directly observe how differing circuit states impact host gene expression and the burdens imposed by the circuits. Such analysis, however, is crucially dependent on the availability of highly pure, intact RNA isolated from fresh or frozen cell cultures. RNA extraction from the yeast Pichia pastoris requires specific pretreatment steps to ensure the reproducibility of downstream applications, but current methods and extraction kits are generally adapted for the conventional yeast Saccharomyces cerevisiae, which has a different cell wall composition. We therefore set out to compare the efficacy of two different RNA isolation methods when applied to P. pastoris: (i) phenol/chloroform extraction and (ii) silica spin-column absorption. We compared the yield, integrity, and purity of the resulting isolated RNA from the two methods (using two different types of commercial columns for silica spin-column absorption) and further optimized them through variations in the pretreatment steps. We also assessed two different methods of cell lysis: enzyme catalytic disruption using lyticase and mechanical disruption using acid-washed glass-beads in a TissueLyser. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: RNA isolation with phenol/chloroform extraction: monophasic lysis reagentAlternate Protocol 1: RNA isolation with silica-spin column absorption: High Pure RNA Isolation Kit (Roche Life Science)Alternate Protocol 2: RNA isolation with silica-spin column absorption: RNeasy Mini Kit (Qiagen)","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"98 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.101","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42895559","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

NMR Spectroscopy–Based Metabolic Profiling of Biospecimens 基于核磁共振光谱的生物标本代谢谱分析

Current Protocols in Protein Science Pub Date : 2019-12-01 DOI: 10.1002/cpps.98

Arjun Sengupta, A. Weljie

{"title":"NMR Spectroscopy–Based Metabolic Profiling of Biospecimens","authors":"Arjun Sengupta, A. Weljie","doi":"10.1002/cpps.98","DOIUrl":"https://doi.org/10.1002/cpps.98","url":null,"abstract":"Metabolomics refers to study of metabolites in biospecimens such as blood serum, tissues, and urine. Nuclear magnetic resonance (NMR) spectroscopy and ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS; mass spectrometry coupled with liquid chromatography) are most frequently employed to analyze complex biological/clinical samples. NMR is a relatively insensitive tool compared to UPLC‐MS/MS but offers straightforward quantification and identification and easy sample processing. One‐dimensional 1H NMR spectroscopy is inherently quantitative and can be readily used for metabolite quantification without individual metabolite standards. Two‐dimensional spectroscopy is most commonly used for identification of metabolites but can also be used quantitatively. Although NMR experiments are unbiased regarding the chemical nature of the analyte, it is crucial to adhere to the proper metabolite extraction protocol for optimum results. Selection and implementation of appropriate NMR pulse programs are also important. Finally, employment of the correct metabolite quantification strategy is crucial as well. In this unit, step‐by‐step guidance for running an NMR metabolomics experiment from typical biospecimens is presented. The unit describes an optimized metabolite extraction protocol, followed by implementation of NMR experiments and quantification strategies using the so‐called “targeted profiling” technique. This approach relies on an underlying basis set of metabolite spectra acquired under similar conditions. Some strategies for statistical analysis of the data are also presented. Overall, this set of protocols should serve as a guide for anyone who wishes to enter the world of NMR‐based metabolomics analysis. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.98","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46646867","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: 7

Gram‐Scale Preparation of C‐Terminal‐Modified Enkephalin Analogues by Typical Liquid‐Phase Peptide Synthesis 典型液相肽合成法制备C -末端修饰脑啡肽类似物的克级研究

Current Protocols in Protein Science Pub Date : 2019-12-01 DOI: 10.1002/cpps.97

Y. Lee

引用次数: 4

Antibody Panel Based N‐Glycan Imaging for N‐Glycoprotein Biomarker Discovery 基于抗体面板的N -糖蛋白成像用于发现N -糖蛋白生物标志物

Current Protocols in Protein Science Pub Date : 2019-12-01 DOI: 10.1002/cpps.99

Alyson P. Black, P. Angel, Richard R Drake, A. Mehta

{"title":"Antibody Panel Based N‐Glycan Imaging for N‐Glycoprotein Biomarker Discovery","authors":"Alyson P. Black, P. Angel, Richard R Drake, A. Mehta","doi":"10.1002/cpps.99","DOIUrl":"https://doi.org/10.1002/cpps.99","url":null,"abstract":"Antibody panel based N‐glycan imaging is a novel platform for N‐glycan analysis of immunocaptured proteins. N‐glycosylation is a post‐translational modification of pathophysiological importance and is often studied in the context of disease biomarkers. Determination of protein‐specific N‐glycosylation changes in patient samples has traditionally been laborious or limited to study of a single protein per analysis. This novel technique allows for the multiplexed analysis of N‐glycoproteins from biofluids. Briefly, this platform consists of antibodies spotted in an array panel to a microscope slide, specific capture of glycoproteins from a biological sample, and then enzymatic release of N‐glycans for analysis by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS). N‐glycans are detected at each individual spot, allowing N‐glycan information to easily be linked back to its protein carrier. Using this protocol, multiplexed analysis of N‐glycosylation on serum glycoproteins can be performed. Human serum is discussed here, but this method has potential to be applied to other biofluids and to any glycoprotein that can be captured by a validated antibody. © 2019 by John Wiley & Sons, Inc.","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.99","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43093483","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: 15

Preparation of Lipid Nanodiscs with Lipid Mixtures 用脂质混合物制备脂质纳米片

Current Protocols in Protein Science Pub Date : 2019-11-20 DOI: 10.1002/cpps.100

Mavis Jiarong Li, William M. Atkins, Wynton D. McClary

{"title":"Preparation of Lipid Nanodiscs with Lipid Mixtures","authors":"Mavis Jiarong Li, William M. Atkins, Wynton D. McClary","doi":"10.1002/cpps.100","DOIUrl":"10.1002/cpps.100","url":null,"abstract":"Lipid nanodiscs provide a native-like lipid environment for membrane proteins, and they have become a valuable platform for the study of membrane biophysics. A range of biophysical and biochemical analyses are enabled when membrane proteins are captured in lipid nanodiscs. Two parameters that can be controlled when capturing membrane proteins in lipid nanodiscs are the radius, and hence the surface area of the lipid surface, and the composition of the lipid bilayer. Despite their emergence as a versatile tool, most studies with lipid nanodiscs in the literature have focused on nanodiscs of a single radius with a single lipid. In light of the complexity of biological membranes, it is likely that nanodiscs with multiple membrane components would be more sophisticated models for membrane research. It is possible to prepare nanodiscs with more complex lipid mixtures to probe the effects of lipid composition on several aspects of membrane biochemistry. Detailed protocols are described here for the preparation of nanodiscs with mixtures of phospholipids, incorporation of cholesterol, and incorporation of a spectroscopic lipid probe. These protocols provide starting points for the construction of nanodiscs with more physiological membrane compositions or with useful biophysical probes. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: Assembly of mixed lipid nanodiscsBasic Protocol 2: Assembly of nanodiscs with cholesterolBasic Protocol 3: Incorporation of laurdan into nanodiscs for membrane fluidity measurements","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"98 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.100","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41917402","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: 17