Renuk Varayil Lakshmanan, Mavis Agbandje-McKenna, Robert McKenna
{"title":"利用 Intein 技术对 B19V VP1u 进行结构研究的域分割可行性。","authors":"Renuk Varayil Lakshmanan, Mavis Agbandje-McKenna, Robert McKenna","doi":"10.2174/0109298665277211231214065419","DOIUrl":null,"url":null,"abstract":"<p><strong>Introduction: </strong>Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus.</p><p><strong>Objectives: </strong>The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA<sub>2</sub>) domain.</p><p><strong>Methods: </strong>RBD and PLA<sub>2</sub> domains of VP1u were each fused to the DnaE split inteins derived from the <i>Nostoc punctiforme</i>. Each of these precursor proteins was expressed in <i>E. coli</i>. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA<sub>2</sub> assays were used to probe the structure and activity of the newly formed protein.</p><p><strong>Results: </strong>The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA<sub>2</sub> assay indicated minimal disruption in enzymatic activity.</p><p><strong>Conclusion: </strong>This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.\",\"authors\":\"Renuk Varayil Lakshmanan, Mavis Agbandje-McKenna, Robert McKenna\",\"doi\":\"10.2174/0109298665277211231214065419\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Introduction: </strong>Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus.</p><p><strong>Objectives: </strong>The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA<sub>2</sub>) domain.</p><p><strong>Methods: </strong>RBD and PLA<sub>2</sub> domains of VP1u were each fused to the DnaE split inteins derived from the <i>Nostoc punctiforme</i>. Each of these precursor proteins was expressed in <i>E. coli</i>. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA<sub>2</sub> assays were used to probe the structure and activity of the newly formed protein.</p><p><strong>Results: </strong>The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA<sub>2</sub> assay indicated minimal disruption in enzymatic activity.</p><p><strong>Conclusion: </strong>This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.</p>\",\"PeriodicalId\":20736,\"journal\":{\"name\":\"Protein and Peptide Letters\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.0000,\"publicationDate\":\"2024-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Protein and Peptide Letters\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.2174/0109298665277211231214065419\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein and Peptide Letters","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.2174/0109298665277211231214065419","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.
Introduction: Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus.
Objectives: The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2) domain.
Methods: RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein.
Results: The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity.
Conclusion: This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.
期刊介绍：
Protein & Peptide Letters publishes letters, original research papers, mini-reviews and guest edited issues in all important aspects of protein and peptide research, including structural studies, advances in recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, and drug design. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallization and preliminary structure determination of biologically important proteins are considered only if they include significant new approaches or deal with proteins of immediate importance, and preliminary structure determinations of biologically important proteins. Purely theoretical/review papers should provide new insight into the principles of protein/peptide structure and function. Manuscripts describing computational work should include some experimental data to provide confirmation of the results of calculations.
Protein & Peptide Letters focuses on:
Structure Studies
Advances in Recombinant Expression
Drug Design
Chemical Synthesis
Function
Pharmacology
Enzymology
Conformational Analysis
Immunology
Biotechnology
Protein Engineering
Protein Folding
Sequencing
Molecular Recognition
Purification and Analysis
京公网安备 11010802042870号
京ICP备2023020795号-1
分享
求助内容：
应助结果提醒方式：
扫码关注我们
