Hemanthie C. Wickramasinghe , Juliette N. Lincoln , Anne E. D'Armond , Sadie A. Noble , Li Shen , Megan A. Macnaughtan
{"title":"通过在大肠杆菌中的连续表达揭示沙眼衣原体 III 型分泌伴侣复合体 Scc4:Scc1 的关联。","authors":"Hemanthie C. Wickramasinghe , Juliette N. Lincoln , Anne E. D'Armond , Sadie A. Noble , Li Shen , Megan A. Macnaughtan","doi":"10.1016/j.pep.2024.106532","DOIUrl":null,"url":null,"abstract":"<div><p><em>Chlamydia trachomatis</em> (<em>CT</em>) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate, intracellular Gram-negative bacterium has a type III secretion system (T3SS) to invade host cells. CopN is an important effector, plug protein that mediates early interactions between the host and <em>Chlamydia</em>. CopN is chaperoned by a heterodimer, T3SS chaperone complex containing Scc4 and Scc1. Scc4 is a unique, bifunctional protein that, in addition to its T3SS chaperone activity, acts as an RNA polymerase (RNAP) binding protein. We hypothesized that the two functions occur at different points in <em>CT</em>'s developmental cycle with Scc4 acting alone in the early-to-mid stages and the Scc4:Scc1 complex chaperoning CopN in the mid-to-late stages. To study the Scc4:Scc1 complex by NMR, we previously explored various methods of associating Scc4 and Scc1 <em>in vitro</em> to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the <em>in vitro</em> association studies suggest that partial protein denaturation and/or components in <em>E. coli</em> lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in <em>E. coli</em> under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation <em>in vivo</em>. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous <em>in vitro</em> association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in <em>E. coli</em>.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106532"},"PeriodicalIF":1.4000,"publicationDate":"2024-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Insights into the association of the Chlamydia trachomatis type III secretion chaperone complex, Scc4:Scc1, from sequential expression in Escherichia coli\",\"authors\":\"Hemanthie C. Wickramasinghe , Juliette N. Lincoln , Anne E. D'Armond , Sadie A. Noble , Li Shen , Megan A. Macnaughtan\",\"doi\":\"10.1016/j.pep.2024.106532\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p><em>Chlamydia trachomatis</em> (<em>CT</em>) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate, intracellular Gram-negative bacterium has a type III secretion system (T3SS) to invade host cells. CopN is an important effector, plug protein that mediates early interactions between the host and <em>Chlamydia</em>. CopN is chaperoned by a heterodimer, T3SS chaperone complex containing Scc4 and Scc1. Scc4 is a unique, bifunctional protein that, in addition to its T3SS chaperone activity, acts as an RNA polymerase (RNAP) binding protein. We hypothesized that the two functions occur at different points in <em>CT</em>'s developmental cycle with Scc4 acting alone in the early-to-mid stages and the Scc4:Scc1 complex chaperoning CopN in the mid-to-late stages. To study the Scc4:Scc1 complex by NMR, we previously explored various methods of associating Scc4 and Scc1 <em>in vitro</em> to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the <em>in vitro</em> association studies suggest that partial protein denaturation and/or components in <em>E. coli</em> lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in <em>E. coli</em> under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation <em>in vivo</em>. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous <em>in vitro</em> association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in <em>E. coli</em>.</p></div>\",\"PeriodicalId\":20757,\"journal\":{\"name\":\"Protein expression and purification\",\"volume\":\"222 \",\"pages\":\"Article 106532\"},\"PeriodicalIF\":1.4000,\"publicationDate\":\"2024-06-08\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Protein expression and purification\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1046592824001049\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein expression and purification","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1046592824001049","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Insights into the association of the Chlamydia trachomatis type III secretion chaperone complex, Scc4:Scc1, from sequential expression in Escherichia coli
Chlamydia trachomatis (CT) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate, intracellular Gram-negative bacterium has a type III secretion system (T3SS) to invade host cells. CopN is an important effector, plug protein that mediates early interactions between the host and Chlamydia. CopN is chaperoned by a heterodimer, T3SS chaperone complex containing Scc4 and Scc1. Scc4 is a unique, bifunctional protein that, in addition to its T3SS chaperone activity, acts as an RNA polymerase (RNAP) binding protein. We hypothesized that the two functions occur at different points in CT's developmental cycle with Scc4 acting alone in the early-to-mid stages and the Scc4:Scc1 complex chaperoning CopN in the mid-to-late stages. To study the Scc4:Scc1 complex by NMR, we previously explored various methods of associating Scc4 and Scc1 in vitro to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the in vitro association studies suggest that partial protein denaturation and/or components in E. coli lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in E. coli under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation in vivo. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous in vitro association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in E. coli.
期刊介绍:
Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.