Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2
Аутори
Đukić, TeodoraVasović, Tamara
Mladenović, Maja
Jovanović, Vesna B.
Smiljanić, Katarina
Radosavljević, Jelena
Stanić-Vučinić, Dragana
Ćirković-Veličković, Tanja
Конференцијски прилог (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
Nucleocapsid (N) protein is the most abundant SARS-CoV-2 virus derived protein and strong immunogen which can be used as a component of the immunological tests for the diagnosis of SARS-CoV-2 infection. Recombinant fragment of N-protein (58–419 aa) was expressed in E. coli in a soluble form using developed optimized protocol of expression (16-18h, 37 °C, 0.4 mM IPTG). After lysis of cells, N-protein from soluble fraction of lysate was purified using optimized protocol for purification by immobilized metal affinity chromatography on Ni-Sepharose in two repeated steps under different elution conditions. Obtained fraction of N-protein after the second chromatography was desalted and concentrated using phosphate buffer solution and ultrafiltration. The purity of isolated N-protein was deterrmined by SDS PAGE, while high resolution mass spectrometry was used for its characterization. Isolated N-protein was over 90% purity and identified as the most intense and abundant protein fragment, wit...h PEAKS PTM score of 508 and sequence coverage of over 70%, including 173 unique peptides.
Кључне речи:
Nucleocaspid protein / recombinant fragment / E coli / unique peptides / mass spectrometryИзвор:
Serbian Biochemical Society Tenth Conference with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021, 2021, 108-108Издавач:
- Belgrade : Serbian Biochemical Society
Финансирање / пројекти:
- CAPSIDO – Developement of the assays for detection of SARS Cov-2 virus capsid proteins in biological fluids of COVID19 patients (RS-ScienceFundRS-Fond_2020_COVID19-7542203)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200168 (Универзитет у Београду, Хемијски факултет) (RS-MESTD-inst-2020-200168)
Колекције
Институција/група
Hemijski fakultet / Faculty of ChemistryTY - CONF AU - Đukić, Teodora AU - Vasović, Tamara AU - Mladenović, Maja AU - Jovanović, Vesna B. AU - Smiljanić, Katarina AU - Radosavljević, Jelena AU - Stanić-Vučinić, Dragana AU - Ćirković-Veličković, Tanja PY - 2021 UR - http://cherry.chem.bg.ac.rs/handle/123456789/5774 AB - Nucleocapsid (N) protein is the most abundant SARS-CoV-2 virus derived protein and strong immunogen which can be used as a component of the immunological tests for the diagnosis of SARS-CoV-2 infection. Recombinant fragment of N-protein (58–419 aa) was expressed in E. coli in a soluble form using developed optimized protocol of expression (16-18h, 37 °C, 0.4 mM IPTG). After lysis of cells, N-protein from soluble fraction of lysate was purified using optimized protocol for purification by immobilized metal affinity chromatography on Ni-Sepharose in two repeated steps under different elution conditions. Obtained fraction of N-protein after the second chromatography was desalted and concentrated using phosphate buffer solution and ultrafiltration. The purity of isolated N-protein was deterrmined by SDS PAGE, while high resolution mass spectrometry was used for its characterization. Isolated N-protein was over 90% purity and identified as the most intense and abundant protein fragment, with PEAKS PTM score of 508 and sequence coverage of over 70%, including 173 unique peptides. PB - Belgrade : Serbian Biochemical Society C3 - Serbian Biochemical Society Tenth Conference with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021 T1 - Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2 SP - 108 EP - 108 UR - https://hdl.handle.net/21.15107/rcub_cherry_5774 ER -
@conference{ author = "Đukić, Teodora and Vasović, Tamara and Mladenović, Maja and Jovanović, Vesna B. and Smiljanić, Katarina and Radosavljević, Jelena and Stanić-Vučinić, Dragana and Ćirković-Veličković, Tanja", year = "2021", abstract = "Nucleocapsid (N) protein is the most abundant SARS-CoV-2 virus derived protein and strong immunogen which can be used as a component of the immunological tests for the diagnosis of SARS-CoV-2 infection. Recombinant fragment of N-protein (58–419 aa) was expressed in E. coli in a soluble form using developed optimized protocol of expression (16-18h, 37 °C, 0.4 mM IPTG). After lysis of cells, N-protein from soluble fraction of lysate was purified using optimized protocol for purification by immobilized metal affinity chromatography on Ni-Sepharose in two repeated steps under different elution conditions. Obtained fraction of N-protein after the second chromatography was desalted and concentrated using phosphate buffer solution and ultrafiltration. The purity of isolated N-protein was deterrmined by SDS PAGE, while high resolution mass spectrometry was used for its characterization. Isolated N-protein was over 90% purity and identified as the most intense and abundant protein fragment, with PEAKS PTM score of 508 and sequence coverage of over 70%, including 173 unique peptides.", publisher = "Belgrade : Serbian Biochemical Society", journal = "Serbian Biochemical Society Tenth Conference with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021", title = "Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2", pages = "108-108", url = "https://hdl.handle.net/21.15107/rcub_cherry_5774" }
Đukić, T., Vasović, T., Mladenović, M., Jovanović, V. B., Smiljanić, K., Radosavljević, J., Stanić-Vučinić, D.,& Ćirković-Veličković, T.. (2021). Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2. in Serbian Biochemical Society Tenth Conference with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021 Belgrade : Serbian Biochemical Society., 108-108. https://hdl.handle.net/21.15107/rcub_cherry_5774
Đukić T, Vasović T, Mladenović M, Jovanović VB, Smiljanić K, Radosavljević J, Stanić-Vučinić D, Ćirković-Veličković T. Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2. in Serbian Biochemical Society Tenth Conference with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021. 2021;:108-108. https://hdl.handle.net/21.15107/rcub_cherry_5774 .
Đukić, Teodora, Vasović, Tamara, Mladenović, Maja, Jovanović, Vesna B., Smiljanić, Katarina, Radosavljević, Jelena, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, "Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2" in Serbian Biochemical Society Tenth Conference with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021 (2021):108-108, https://hdl.handle.net/21.15107/rcub_cherry_5774 .