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http://hdl.handle.net/11375/29258
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DC Field | Value | Language |
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dc.contributor.advisor | Capone, John P. | - |
dc.contributor.author | Donaldson, Logan William Frederick | - |
dc.date.accessioned | 2023-12-04T16:12:38Z | - |
dc.date.available | 2023-12-04T16:12:38Z | - |
dc.date.issued | 1992-09 | - |
dc.identifier.uri | http://hdl.handle.net/11375/29258 | - |
dc.description.abstract | In order to facilitate a biophysical analysis of the carboxy terminal acidic transactivation domain (AAD) of Vmw65 from Herpes Simplex Virus Type 1 (HSV-1), an overexpression system in Escherichia coli was constructed and optimized to produce milligram quantities of this polypeptide. Purification of the polypeptide was facilitated by creating a fusion protein to glutathione S-transferase (GST) from Schizosoma japonicum using a commercially available vector. Upon thrombin digestion of the fusion protein, the carrier and AAD products were resolved by anion-exchange chromatography. With typically 15 mg of AAD available from a 12 litre culture, several biophysical studies were initiated. Circular dichroism and fluorescence spectroscopy both described a polypeptide with an extended structure reminicent of a random-coil; that is, it did not possess substantial quantities of known elements of secondary structure such as a-helicies and β-sheets under physiological conditions. A new structure high in α-helical content was induced upon addition of trifluoroethanol to mimic a hydrophobic milieu. Ultracentrifugation data supported the spectroscopic observations by describing an extended, monomeric polypeptide. The ultimate goal of the study, a teritiary structure, was sought by attempting to crystallize AAD with popular salts and organic solvents. Biologically, the described random-coil structure of AAD could be relevant to its role as a promoter and stablizer of the transcriptional pre-initation complex, the determining step in gene expression. A structurally labile domain would support AAD’s ability to interact with several targets including TFIID and TFIIB, though not necessarily by similar mechanisms. The requirement for a drastic conformational change such as a random-coil to α-helical transition currently remains unclear though observations made in this study of AAD in trifluoroethanol have shown that a conformational change is indeed possible. With a means of producing large quantities of AAD, the opportunity now arises to study its interaction with available cloned targets. The ensuing biophysical studies will then provide a greater understanding of AAD’s important role in gene expression. | en_US |
dc.language.iso | en | en_US |
dc.subject | Herpes Simplex Virus Type 1 | en_US |
dc.subject | Carboxy Terminal Transactivation Domain | en_US |
dc.title | Overexpression, Purification and Biophysical Studies of the Carboxy Terminal Transactivation Domain of Vmw65 from Herpes Simplex Virus Type 1 | en_US |
dc.type | Thesis | en_US |
dc.contributor.department | Biochemistry | en_US |
dc.description.degreetype | Thesis | en_US |
dc.description.degree | Master of Science (MSc) | en_US |
Appears in Collections: | Digitized Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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donaldson_logan_w_f_1992Sept_masters.pdf | 19.18 MB | Adobe PDF | View/Open |
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