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Thesis
Purification and characterization of a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen DNA-binding domain mutant
Master of Science (M.S.), Drexel University
Apr 2018
DOI:
https://doi.org/10.17918/D8QQ2X
Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) infection is associated with three human malignancies: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and Multicentric Castleman's disease (MCD). Latency-associated nuclear antigen (LANA) is one of the few viral proteins expressed during latent KSHV infection. It acts as the master regulator of KSHV latency and is essential for viral replication and episome maintenance. Previous studies have shown that these functions are promoted by oligomerization of LANA, which is a process mediated by the DNA-binding domain (DBD). The structure of the wild-type LANADBD forms higher-order oligomers upon crystallization, which poses a challenge to characterizing the DBD. Mutations within the dimer-dimer interface of LANA, specifically at residues F1037 and F1041, disrupt higher-order oligomerization. Crystallization of lower-order oligomers could improve resolution of the DBD. Given this, we sought to purify and crystallize the LANA DBD F1037A/F1041A double mutant (LANA,DBD,AA). Bacterial expression of LANADBD,AA was optimized by varying IPTG concentration, the duration of IPTG induction, and the induction temperature. Multiple methods of protein purification were utilized, including batch affinity purification, affinity column chromatography and ion-exchange column chromatography. Purified LANA,DBD,AA was subjected to a variety of crystallization conditions. These conditions included attempts to optimize buffer pH, %PEG, and molecular weight of PEG. Future directions will focus on determining the structure of LANA,DBD,AA, as well as the structures of the single mutants F1037A (LANA,DBD,AF) and F1041 (LANA,DBD,FA). These mutant structures will aid in the design and development of a small molecule that inhibits LANA-DNA binding.
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Details
- Title
- Purification and characterization of a Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen DNA-binding domain mutant
- Creators
- Carlie Sunga Mendoza - DU
- Contributors
- Paul M. Lieberman (Advisor) - Drexel University (1970-)Mauricio Reginato (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Medicine (Graduate); College of Medicine; Hematology and Oncology; Drexel University
- Other Identifier
- 7963; 991014632677604721