Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coli
Globally, tuberculosis (TB) remains a leading cause of death. The emergence of multidrug-resistant strains (MDR-TB) and extensively drug-resistant strains (XDR-TB) has fuelled the discovery for novel drugs and drug targets for its successful and better treatment. One of the potential candidates for...
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| Format: | Article |
| Language: | English |
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Indonesian Society for Microbiology
2015-11-01
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| Series: | Microbiology Indonesia |
| Subjects: | |
| Online Access: | https://jurnal.permi.or.id/index.php/mionline/article/view/321 |
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| author | ERNAWATI ARIFIN GIRI-RACHMAN FENRYCO PRATAMA OKTIRA ROKA AJI ARUM PATRIATI IHSANAWATI IHSANAWATI MAELITA RAMDANI MOEIS EDY GIRI-RACHMAN PUTRA |
| author_facet | ERNAWATI ARIFIN GIRI-RACHMAN FENRYCO PRATAMA OKTIRA ROKA AJI ARUM PATRIATI IHSANAWATI IHSANAWATI MAELITA RAMDANI MOEIS EDY GIRI-RACHMAN PUTRA |
| author_sort | ERNAWATI ARIFIN GIRI-RACHMAN |
| collection | DOAJ |
| description | Globally, tuberculosis (TB) remains a leading cause of death. The emergence of multidrug-resistant strains (MDR-TB) and extensively drug-resistant strains (XDR-TB) has fuelled the discovery for novel drugs and drug targets for its successful and better treatment. One of the potential candidates for drug target is PhoR sensory protein histidine kinase, a part of the Two Component System (TCS) PhoP/PhoR in Mycobacterium tuberculosis (Mtb). This protein system was known for its role on regulating hundred of Mtb virulence factors, from genes for cell wall and lypid synthesis to genes for adaptation in human leukocyte and hypoxia response. Previous studies have successfully characterized, isolated, and cloned the putative sensory domain of PhoR protein gene into pRSET vector expression system. In this study, Escherichia coli was transformed with pRSET-SensPhoR and cultivated at 37oC under IPTG induction to express PhoR sensor-domain protein. Most of the proteins were overexpressed in the form of inclusion bodies. Subsequent protein purification in Ni-NTA system under refolding condition on urea gradient was performed to isolate PhoR sensor-domain protein in soluble form. Arginine was supplemented in purified protein solution to prevent aggregation during long term storage. While highly purified protein was acquired, small angle X-ray scattering (SAXS) analysis was conducted to obtain 3-dimensional (3D) protein structures in solution.
doi:10.5454/mi.9.2.1 |
| first_indexed | 2024-12-14T02:31:24Z |
| format | Article |
| id | doaj.art-bd86cbb320db42ab88df02d68f3a2036 |
| institution | Directory Open Access Journal |
| issn | 1978-3477 2087-8575 |
| language | English |
| last_indexed | 2024-12-14T02:31:24Z |
| publishDate | 2015-11-01 |
| publisher | Indonesian Society for Microbiology |
| record_format | Article |
| series | Microbiology Indonesia |
| spelling | doaj.art-bd86cbb320db42ab88df02d68f3a20362022-12-21T23:20:15ZengIndonesian Society for MicrobiologyMicrobiology Indonesia1978-34772087-85752015-11-0192Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coliERNAWATI ARIFIN GIRI-RACHMANFENRYCO PRATAMAOKTIRA ROKA AJIARUM PATRIATIIHSANAWATI IHSANAWATIMAELITA RAMDANI MOEISEDY GIRI-RACHMAN PUTRAGlobally, tuberculosis (TB) remains a leading cause of death. The emergence of multidrug-resistant strains (MDR-TB) and extensively drug-resistant strains (XDR-TB) has fuelled the discovery for novel drugs and drug targets for its successful and better treatment. One of the potential candidates for drug target is PhoR sensory protein histidine kinase, a part of the Two Component System (TCS) PhoP/PhoR in Mycobacterium tuberculosis (Mtb). This protein system was known for its role on regulating hundred of Mtb virulence factors, from genes for cell wall and lypid synthesis to genes for adaptation in human leukocyte and hypoxia response. Previous studies have successfully characterized, isolated, and cloned the putative sensory domain of PhoR protein gene into pRSET vector expression system. In this study, Escherichia coli was transformed with pRSET-SensPhoR and cultivated at 37oC under IPTG induction to express PhoR sensor-domain protein. Most of the proteins were overexpressed in the form of inclusion bodies. Subsequent protein purification in Ni-NTA system under refolding condition on urea gradient was performed to isolate PhoR sensor-domain protein in soluble form. Arginine was supplemented in purified protein solution to prevent aggregation during long term storage. While highly purified protein was acquired, small angle X-ray scattering (SAXS) analysis was conducted to obtain 3-dimensional (3D) protein structures in solution. doi:10.5454/mi.9.2.1https://jurnal.permi.or.id/index.php/mionline/article/view/321Tuberculosismulti-drug resistancehistidine kinase componentrational drug design |
| spellingShingle | ERNAWATI ARIFIN GIRI-RACHMAN FENRYCO PRATAMA OKTIRA ROKA AJI ARUM PATRIATI IHSANAWATI IHSANAWATI MAELITA RAMDANI MOEIS EDY GIRI-RACHMAN PUTRA Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coli Microbiology Indonesia Tuberculosis multi-drug resistance histidine kinase component rational drug design |
| title | Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coli |
| title_full | Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coli |
| title_fullStr | Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coli |
| title_full_unstemmed | Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coli |
| title_short | Expression and Purification of PhoR Sensor-Domain Histidine Kinase of Mycobacterium tuberculosis in Escherichia coli |
| title_sort | expression and purification of phor sensor domain histidine kinase of mycobacterium tuberculosis in escherichia coli |
| topic | Tuberculosis multi-drug resistance histidine kinase component rational drug design |
| url | https://jurnal.permi.or.id/index.php/mionline/article/view/321 |
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