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Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2005 (English)In: Nature Methods, ISSN 1548-7091, Vol. 2, no 7, 507-509 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2005. Vol. 2, no 7, 507-509 p.
Identifiers
URN: urn:nbn:se:su:diva-25545OAI: oai:DiVA.org:su-25545DiVA: diva2:199932
Note
Part of urn:nbn:se:su:diva-8261Available from: 2008-10-23 Created: 2008-10-14 Last updated: 2010-01-13Bibliographically approved
In thesis
1. Structural Studies on PP2A and Methods in Protein Production
Open this publication in new window or tab >>Structural Studies on PP2A and Methods in Protein Production
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

PP2A is a major phosphatase in the cell that participates in multiple cell signaling pathways. It is a heterotrimer of a core dimer and variable regulatory subunits. Details of its structure, function and regulation are slowly emerging. Here, the structure of two regulators of PP2A are de-scribed; PTPA and B56γ. PTPA is a highly conserved enzyme that plays a crucial role in PP2A activity but whose biochemical function is still unclear. B56γ is a PP2A regulatory subunit linked to cancer and the structure presented here of B56γ in its free form is particularly valuable in light of the recent structures of the PP2A holoenzyme and core dimer.

Protein production is a major bottleneck in structural genomic projects. Here, we describe two novel methods for improved protein production. The first is a colony based screening method where any DNA library can be screened for soluble expression of recombinant proteins in E.coli. The second method involves improvements of the well established IMAC purification method. We have seen that a low molecular weight component of E.coli lysate decreases the binding capacity of IMAC columns and by removing the low molecular weight components, recombinant proteins only present at low levels in E.coli lysate can be purified, which has previously been believed to be unfeasible.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 53 p.
Keyword
PP2A, PTPA, B56γ, Protein Production
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-8261 (URN)978-91-7155-750 (ISBN)
Public defence
2008-11-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-10-23 Created: 2008-10-14Bibliographically approved
2. Structural studies of proteins in apoptosis and lipid signaling
Open this publication in new window or tab >>Structural studies of proteins in apoptosis and lipid signaling
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Signaling pathways control the fate of the cell. For example, they promote cell survival or commit the cell to death (apoptosis) in response to cell injury or developmental stimuli, decisions, which are vital for the proper development and functioning of metazoan. Tight control of such pathways is essential; dysregulation of apoptosis can disrupt the delicate balance between cell proliferation and cell death ending up in pathological processes, including cancer, autoimmunity diseases, inflammatory diseases, or degenerative disorders. We have used a structural genomic approach to study the structure and function of key proteins involved in apoptosis and lipid signaling: the antiapoptotic Bcl-2 family member Bfl-1 in complex with a Bim peptide, the BIR domains of the Inhibitor of Apoptosis (IAP) family members, cIAP2 and NAIP and the a lipid kinase YegS. The structural analysis of the apoptosis regulatory proteins has revealed important information on the structural determinants for recognition of interacting proteins, which can now assist in the development of therapeutic drugs for human diseases. The structural and complementing biochemical studies of the lipid kinase YegS have reveled the first detailed information on a lipid kinase and explained important aspects of its structure-function relationship.

Finally, one subject of this work aim to solve what is arguably the most challenging problem in structural projects – to obtain a high production level of proteins suitable for structural studies. We have developed a highthroughput protein solubility screening, the colony filtration (CoFi) blot, which allows soluble clones to be identified from large libraries of protein variants and now constitute a powerful tool for solving difficult protein production problems.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 74 p.
Keyword
proteins, crystal structure, apoptosis, cancer, CoFi-Blot, kinases, lipid signaling
National Category
Structural Biology
Research subject
Structural Biology
Identifiers
urn:nbn:se:su:diva-8212 (URN)978-91-7155-703-2 (ISBN)
Public defence
2008-09-11, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2008-09-22 Created: 2008-09-22 Last updated: 2010-01-13Bibliographically approved
3. Expression and structure-function characterisation of herpesviral proteins
Open this publication in new window or tab >>Expression and structure-function characterisation of herpesviral proteins
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
Protein production, Herpesvirus, SOX, E. coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-8288 (URN)978-91-7155-755-1 (ISBN)
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
4. Novel Technologies for Recombinant Protein Overexpression in Escherichia coli
Open this publication in new window or tab >>Novel Technologies for Recombinant Protein Overexpression in Escherichia coli
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of recombinant protein is a cornerstone in many structural and functional studies. The enteric bacterium Escherichia Coli is the most commonly used organism for producing recombinant proteins. E. coli has several advantages over other expression hosts, but also one major disadvantage - the protein of interest does not always adopt its native conformation. Instead the protein might form large insoluble aggregates, inclusion bodies, within the cell. In particular, the heterologous overexpression of eukaryotic and membrane proteins are troublesome.

In this thesis, methods are described that can be used to increase the likelihood of overexpressing eukaryotic proteins as well as membrane proteins. In particular, a novel method is described that can distinguish between bacterial colonies expressing soluble proteins from those expressing inclusion bodies. The method utilizes the fact that inclusion bodies are of a considerable size and can be removed by filtration. Using this screening method in combination with methods that alter the physical properties of proteins, we have shown that the likelihood of overexpression in E. coli can be dramatically increased.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2006. 76 p.
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-861 (URN)91-7155-212-X (ISBN)
Public defence
2006-03-24, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2006-03-03 Created: 2006-03-03 Last updated: 2010-01-13Bibliographically approved

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