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Structure and Functional Study on HigBA from Streptococcus pneumoniae
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 이봉진 | - |
dc.contributor.author | 김성룡 | - |
dc.date.accessioned | 2017-10-31T08:19:48Z | - |
dc.date.available | 2020-10-06T08:35:04Z | - |
dc.date.issued | 2017-08 | - |
dc.identifier.other | 000000146083 | - |
dc.identifier.uri | https://hdl.handle.net/10371/137935 | - |
dc.description | 학위논문 (석사)-- 서울대학교 대학원 약학대학 약학과, 2017. 8. 이봉진. | - |
dc.description.abstract | Streptococcus pneumoniae is a gram-positive strain that causes
diseases mainly through respiratory infections. Diseases caused by pneumococcal species are meningitis, bacteremia, pneumonia, otitis and sinusitis. Patients infected with s.pneumonia have developed antibiotic resistant strains, so new antibiotics need to be developed. Ultimately, developing novel antibiotic candidates through structural analysis and functional studies of proteins is a major goal. The target TA complex protein present in the s.pneumoniae TIGR4 strain is a type II toxin-antitoxin system and is classified under HigBA family. The toxin protein HigB is predicted to be a ribosome-dependent mRNA interferases, which cleave mRNAs at the ribosomal A site. The antitoxin protein HigA regulates transcription through binding of palindromic sequence to its operator region. In order to obtain the tertiary structure of the protein, the target protein was obtained by recombinant process and was over-expressed in Rosetta (DE3) pLysS of escherichia coli. Affinity chromatography was used to purify the hexa-histidine tagged protein. Further, higher purity of target protein was obtained by ion-exchange chromatography and size-exclusion chromatography. The structure of target protein was obtained using X-ray crystallography techniques. | - |
dc.description.tableofcontents | I. Introduction ····························································· 1
1.1. Streptococcus pneumoniae ···································· 1 1.2. Toxin-Antitoxin system ·········································· 2 1.3. HigBA family in type II TA system ··············· 3 1.4. Purpose of Study ························································ 4 II. Materials and Methods ····································· 5 2.1. Materials ········································································· 5 2.1.1. Reagents ············································································5 2.1.2. Apparatus ··········································································5 2.2. Methods ··········································································· 6 2.2.1. Gene cloning ·····································································6 2.2.2. Over-expression and purification ································7 2.2.3. Crystallization ···································································8 2.2.4. X-ray data collection and structure determination ···············································8 2.2.5. EMSA (Electrophoretic Mobility Shift Assay) ······· 9 2.2.6. Ribonuclease activity assay ··········································9 III. Results ·································································· 11 3.1. Over-expression and purification ······················ 11 3.1.1. Over expression ·······························································11 3.1.2. Purification ········································································12 3.1.2.1. Affinity chromatography ········································12 3.1.2.2. Ion exchange chromatography ······························12 3.1.2.3. Size exclusion chromatography ····························13 3.2. Crystallization ····························································· 14 3.3. EMSA (Electrophoretic Mobility Shift Assay) ····························································· 15 3.4. I n vitro ribonuclease activity assay ················· 17 V. Discussion ····························································· 19 VI. References ··························································· 23 국문초록 ········································································ 28 | - |
dc.format | application/pdf | - |
dc.format.extent | 699345 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Streptococcus pneumoniae | - |
dc.subject | Toxin-antitoxin system | - |
dc.subject | HigBA | - |
dc.subject | X-ray crystallography | - |
dc.subject | EMSA | - |
dc.subject | RNase activity assay | - |
dc.subject.ddc | 615 | - |
dc.title | Structure and Functional Study on HigBA from Streptococcus pneumoniae | - |
dc.type | Thesis | - |
dc.description.degree | Master | - |
dc.contributor.affiliation | 약학대학 약학과 | - |
dc.date.awarded | 2017-08 | - |
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