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A NOVEL IN-CELL LETHALITY-BASED MOLECULAR SCREENING SYSTEM
USING A SPLIT BARNASE
by
Jie Zhou
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2012
Copyright 2012 Jie Zhou
Object Description
| Title | A novel in-cell lethality-based molecular screening system using a split barnase |
| Author | Zhou, Jie |
| Author email | sarah.jzhou.usc@gmail.com |
| Degree | Master of Science |
| Document type | Dissertation |
| Degree program | Pharmaceutical Sciences |
| School | School of Pharmacy |
| Date defended/completed | 2012-07-31 |
| Date submitted | 2012-07-31 |
| Date approved | 2012-08-01 |
| Restricted until | 2012-08-01 |
| Date published | 2012-08-01 |
| Advisor (committee chair) | Camarero, Julio A. |
| Advisor (committee member) |
Shen, Wei-Chiang Okamoto, Curtis Toshio |
| Abstract | We have developed a cell-based reporter assay using the cytotoxic ribonuclease barnase aimed at identifying new protein-protein inhibitors. Barnase is an extensively well-characterized N1/T1 ribonuclease, which can hydrolyze RNA, inhibit protein synthesis and efficiently trigger cell death. We have used the FK506-binding protein (FKBP)-rapamycin-FKBP12-rapamycin (Rap)-binding protein (FRB) protein complex as a model system to test the feasibility and efficiency of this new cell-based reporter. Barnase is expressed two fragments, N-barnase (NBn) and C-barnase (Cbn), which are linked to the model proteins FKBP and FRB, respectively. In the absence of rapamycin, FKBP and FRB have a relatively low affinity to each other. The split barnase fragments NBn and CBn have also very low affinity for each other and remain inactive. Addition of rapamycin induces the formation fo the complex FKBP-Rap-FRB, which brings in close proximity the barnase fragments allowing them to fold in trans into an active barnase. In-cell activation of barnase activity rapidly triggers cell death. To optimize the split barnase reporter five mutants were designed to completely reduce the self-asscoation between the NBn and CBn fragments in the absence of Rap. These included FRB-CBn-R110A, FRB-CBn-R110D, FRB-CBn-H102Q, FKBP-NBn-D8N, FKBP-NBn-D8R. All these mutations were introduced onto the barnase fragments to detroy potential ionic intereactions between the two brnase fragments. The barnase activity of the different FRB-CBn/FKBP-NBn split barnase pairs was tested in vitro in the absence and presence of Rap.Among all pairs, the pair of FRB-CBn-WT and FKBP-NBn-D8R and the pair of FRB-CBn-R110A and FKBP-NBn-WT showed the highest activity/background ratio, and thus form the ideal pairs for inhibition-drug screening in vitro. Future work will involve the co-expression of the optimized split barnase constructs in Escherichia coli cells to further test the feasibility of this system in live cells. |
| Keyword | Barnase; lethality; screening system; protein interaction inhibitor |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Rights | Zhou, Jie |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume4/etd-ZhouJie-1090.pdf |
Description
| Title | Page 1 |
| Full text | A NOVEL IN-CELL LETHALITY-BASED MOLECULAR SCREENING SYSTEM USING A SPLIT BARNASE by Jie Zhou A Thesis Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (PHARMACEUTICAL SCIENCES) August 2012 Copyright 2012 Jie Zhou |
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