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Expression and structure-function characterisation of herpesviral proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Responsible organisation
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In order to determine and study a protein structure, large amounts of it is needed. The easiest way to obtain a protein is to recombinantly overexpress it in the well-studied bacterium Escherichia coli. However, this expression host has one major disadvantage, overexpressed proteins might not be folded or be insoluble. Within the field of structural genomics, protein production has become one of the most challenging problems and the recombinant overexpression of viral proteins has in particular proven to be difficult.

The first part of the thesis concerns the recombinant overexpression of troublesome proteins in E. coli. A method has been developed to screen for soluble overexpression in E. coli at the colony level, making it suitable for screening large gene collections. This method was used to successfully screen deletion libraries of difficult mammalian proteins as well as ORFeomes from five herpesviruses. As a result soluble expression of previously insoluble mammalian proteins was obtained as well as crystals of three proteins from two oncogenic human herpesviruses, all linked to DNA synthesis of the viral genome. The second part of the work presented concerns the structural studies of three herpesviral proteins. SOX from Kaposi’s sarcoma associated herpesvirus is involved in processing and maturation of the viral genome. Recently SOX has also been implicated in host shutoff at the mRNA level. With this structure, we propose a substrate binding site and a likely exonucleolytic mechanism. The holoenzyme ribonucleotide reductase is solely responsible for the production of deoxyribonucleotides and regulates the nucleotide pool of the cell. The small subunit, R2, has been solved from both Epstein Barr virus and KSHV. Both structures show disordered secondary structure elements in their apo-and mono metal forms, located close to the iron binding sites in similarity to the p53 induced R2 indicating that these two R2 proteins might play a similar and important role.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik , 2008. , 76 p.
Keyword [en]
Protein production, Herpesvirus, SOX, E. coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-8288ISBN: 978-91-7155-755-1 (print)OAI: oai:DiVA.org:su-8288DiVA: diva2:199978
Public defence
2008-11-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 09:00 (English)
Opponent
Supervisors
Available from: 2008-10-30 Created: 2008-10-23 Last updated: 2010-01-13Bibliographically approved
List of papers
1. Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli
Open this publication in new window or tab >>Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli
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2005 (English)In: Nature Methods, ISSN 1548-7091, Vol. 2, no 7, 507-509 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25545 (URN)
Note
Part of urn:nbn:se:su:diva-8261Available from: 2008-10-23 Created: 2008-10-14 Last updated: 2010-01-13Bibliographically approved
2. An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries
Open this publication in new window or tab >>An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries
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2006 (English)In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, Vol. 65, no 2, 266-273 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25544 (URN)
Note
Part of urn:nbn:se:su:diva-8261Available from: 2008-10-23 Created: 2008-10-14 Last updated: 2010-01-13Bibliographically approved
3. Colony filtration blot for screening soluble expression in Escherichia coli
Open this publication in new window or tab >>Colony filtration blot for screening soluble expression in Escherichia coli
2006 In: Nature protocols, ISSN 1754-2189, Vol. 1, no 1, 253-258 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25559 (URN)
Note
Part of urn:nbn:se:su:diva-8288Available from: 2008-10-30 Created: 2008-10-23Bibliographically approved
4. Screening Colonies of Pooled ORFeomes, SCOOP: A rapid and efficient strategy for expression screening ORFeomes in E. coli
Open this publication in new window or tab >>Screening Colonies of Pooled ORFeomes, SCOOP: A rapid and efficient strategy for expression screening ORFeomes in E. coli
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-25560 (URN)
Note
Part of urn:nbn:se:su:diva-8288Available from: 2008-10-30 Created: 2008-10-23 Last updated: 2010-01-13Bibliographically approved
5. Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi's sarcoma-associated herpesvirus
Open this publication in new window or tab >>Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi's sarcoma-associated herpesvirus
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2009 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 22, 6636-6645 p.Article in journal (Refereed) Published
Abstract [en]

The Kaposi's sarcoma-associated herpesvirus protein SOX (shut off and exonuclease) and its Epstein–Barr virus homolog, BGLF5, are active during the early lytic phase and belong to the alkaline nuclease family. Both proteins have been shown to be bifunctional, being responsible for DNA maturation as well as host shutoff at the mRNA level. We present the crystal structure of SOX determined at 1.85 Å resolution. By modeling DNA binding, we have identified catalytic residues that explain the preferred 5'-exonuclease activity of the alkaline nucleases. The presence of a crevice suitable for binding duplex DNA supports a role for herpes alkaline nucleases in recombination events preceding packaging of viral DNA. Direct interaction with dsDNA is supported by oligonucleotide binding data. Mutations specifically affecting host shutoff map to a surface region of the N-terminal domain, implying an essential role in protein–protein interactions, and link the RNase activity of the enzyme to mRNA degradation pathways.

Identifiers
urn:nbn:se:su:diva-25561 (URN)10.1111/j.1742-4658.2009.07374.x (DOI)000271057200021 ()
Note
Part of urn:nbn:se:su:diva-8288Available from: 2008-10-30 Created: 2008-10-23 Last updated: 2017-12-13Bibliographically approved

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