Mia Tria Novianti, Toto Subroto, Yusuf Sofyan Efendi, Umi Baroroh, Shinta Kusumawardani, Gilang Gumilar, Muhammad Yusuf, Shabarni Gaffar
{"title":"在大肠杆菌 BL21(DE3) 中表达的重组白喉类毒素 (rCRM197EK) 的表达、Sarkosyl 溶解、DNase 活性、纯化和 SPR 结合亲和力。","authors":"Mia Tria Novianti, Toto Subroto, Yusuf Sofyan Efendi, Umi Baroroh, Shinta Kusumawardani, Gilang Gumilar, Muhammad Yusuf, Shabarni Gaffar","doi":"10.1007/s12033-024-01238-y","DOIUrl":null,"url":null,"abstract":"<p><p>CRM197EK is a derivative of diphtheria toxoid cross-reactive material-197 (CRM197) with two-point mutations (K51E and E148K) to improve its properties for a vaccine conjugate and drug delivery. A previous study has shown that intracellularly expressing CRM197EK in Escherichia coli (E. coli) host formed inclusion bodies that need a complicated purification and refolding step. Protein purification from inclusion bodies can be overcome by solubilization of inclusion bodies by using N-lauroyl sarcosine (sarkosyl). In this work, recombinant CRM197EK (rCRM197EK) was expressed in E. coli BL21 (DE3) as inclusion bodies, then solubilized using sarkosyl to form a soluble rCRM197EK without the need for a renaturation process. Furthermore, rCRM197EK was purified using the Ni-NTA column, characterized by SDS-PAGE and Western Blot, and its biological activity was assayed through its DNase activity. Moreover, its binding affinity with anti-diphtheria toxin (DT) antibody was measured using the surface plasmon resonance (SPR). The result showed that solubilization with sarkosyl form soluble rCRM197EK (61.61 kDa) was confirmed by SDS-PAGE and Western Blot with a yield of 2.8 mg/mL. rCRM197EK shows DNase activity, and the SPR assay shows that it can interact with an anti-DT antibody with a binding energy of - 9.2 kcal/mol.</p>","PeriodicalId":18865,"journal":{"name":"Molecular Biotechnology","volume":null,"pages":null},"PeriodicalIF":2.4000,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Expression, Sarkosyl Solubilization, DNase Activity, Purification, and SPR Binding Affinity of Recombinant Diphtheria Toxoid (rCRM197EK) Expressed in Escherichia coli BL21(DE3).\",\"authors\":\"Mia Tria Novianti, Toto Subroto, Yusuf Sofyan Efendi, Umi Baroroh, Shinta Kusumawardani, Gilang Gumilar, Muhammad Yusuf, Shabarni Gaffar\",\"doi\":\"10.1007/s12033-024-01238-y\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>CRM197EK is a derivative of diphtheria toxoid cross-reactive material-197 (CRM197) with two-point mutations (K51E and E148K) to improve its properties for a vaccine conjugate and drug delivery. A previous study has shown that intracellularly expressing CRM197EK in Escherichia coli (E. coli) host formed inclusion bodies that need a complicated purification and refolding step. Protein purification from inclusion bodies can be overcome by solubilization of inclusion bodies by using N-lauroyl sarcosine (sarkosyl). In this work, recombinant CRM197EK (rCRM197EK) was expressed in E. coli BL21 (DE3) as inclusion bodies, then solubilized using sarkosyl to form a soluble rCRM197EK without the need for a renaturation process. Furthermore, rCRM197EK was purified using the Ni-NTA column, characterized by SDS-PAGE and Western Blot, and its biological activity was assayed through its DNase activity. Moreover, its binding affinity with anti-diphtheria toxin (DT) antibody was measured using the surface plasmon resonance (SPR). The result showed that solubilization with sarkosyl form soluble rCRM197EK (61.61 kDa) was confirmed by SDS-PAGE and Western Blot with a yield of 2.8 mg/mL. rCRM197EK shows DNase activity, and the SPR assay shows that it can interact with an anti-DT antibody with a binding energy of - 9.2 kcal/mol.</p>\",\"PeriodicalId\":18865,\"journal\":{\"name\":\"Molecular Biotechnology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.4000,\"publicationDate\":\"2024-08-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular Biotechnology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1007/s12033-024-01238-y\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Biotechnology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s12033-024-01238-y","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Expression, Sarkosyl Solubilization, DNase Activity, Purification, and SPR Binding Affinity of Recombinant Diphtheria Toxoid (rCRM197EK) Expressed in Escherichia coli BL21(DE3).
CRM197EK is a derivative of diphtheria toxoid cross-reactive material-197 (CRM197) with two-point mutations (K51E and E148K) to improve its properties for a vaccine conjugate and drug delivery. A previous study has shown that intracellularly expressing CRM197EK in Escherichia coli (E. coli) host formed inclusion bodies that need a complicated purification and refolding step. Protein purification from inclusion bodies can be overcome by solubilization of inclusion bodies by using N-lauroyl sarcosine (sarkosyl). In this work, recombinant CRM197EK (rCRM197EK) was expressed in E. coli BL21 (DE3) as inclusion bodies, then solubilized using sarkosyl to form a soluble rCRM197EK without the need for a renaturation process. Furthermore, rCRM197EK was purified using the Ni-NTA column, characterized by SDS-PAGE and Western Blot, and its biological activity was assayed through its DNase activity. Moreover, its binding affinity with anti-diphtheria toxin (DT) antibody was measured using the surface plasmon resonance (SPR). The result showed that solubilization with sarkosyl form soluble rCRM197EK (61.61 kDa) was confirmed by SDS-PAGE and Western Blot with a yield of 2.8 mg/mL. rCRM197EK shows DNase activity, and the SPR assay shows that it can interact with an anti-DT antibody with a binding energy of - 9.2 kcal/mol.
期刊介绍:
Molecular Biotechnology publishes original research papers on the application of molecular biology to both basic and applied research in the field of biotechnology. Particular areas of interest include the following: stability and expression of cloned gene products, cell transformation, gene cloning systems and the production of recombinant proteins, protein purification and analysis, transgenic species, developmental biology, mutation analysis, the applications of DNA fingerprinting, RNA interference, and PCR technology, microarray technology, proteomics, mass spectrometry, bioinformatics, plant molecular biology, microbial genetics, gene probes and the diagnosis of disease, pharmaceutical and health care products, therapeutic agents, vaccines, gene targeting, gene therapy, stem cell technology and tissue engineering, antisense technology, protein engineering and enzyme technology, monoclonal antibodies, glycobiology and glycomics, and agricultural biotechnology.