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Structural Studies on PP2A and Methods in Protein Production
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Responsible organisation
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 [en]
PP2A, PTPA, B56γ, Protein Production
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-8261ISBN: 978-91-7155-750 OAI: oai:DiVA.org:su-8261DiVA: diva2:199933
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
List of papers
1. The structure of the PP2A regulatory subunit B56gamma: The remaining piece of the PP2A jigsaw puzzle
Open this publication in new window or tab >>The structure of the PP2A regulatory subunit B56gamma: The remaining piece of the PP2A jigsaw puzzle
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2009 (English)In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 74, no 1, 212-221 p.Article in journal (Refereed) Published
Abstract [en]

The PP2A serine/threonine phosphatase regulates a plethora of cellular processes. In the cell the predominant form of the enzyme is a heterotrimer, formed by a core dimer composed of a catalytic and a scaffolding subunit, which assemble together with one of a range of different regulatory B subunits. Here, we present the first structure of a free non-complexed B subunit, B56. Comparison with the recent structures of a heterotrimeric complex and the core dimer reveals several significant conformational changes in the interface region between the B56 and the core dimer. These allow for an assembly scheme of the PP2A holoenzyme to be put forth where B56 first complexes with the scaffolding subunit and subsequently binds to the catalytic subunit and this induces the formation of a binding site for the invariant C-terminus of the catalytic subunit that locks in the complex as a last step of assembly.

Keyword
crystal structure, holoenzyme assembly, PP2A regulation, PR61, B56, B regulatory subunits, protein phosphatase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-25541 (URN)10.1002/prot.22150 (DOI)000261757900019 ()
Available from: 2008-10-23 Created: 2008-10-14 Last updated: 2017-12-13Bibliographically approved
2. IMAC purification has severe and serious limitations: Means for improvement
Open this publication in new window or tab >>IMAC purification has severe and serious limitations: Means for improvement
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-25542 (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. The crystal structure of a human PP2A phosphatase activator reveals a novel fold and highly conserved cleft implicated in protein-protein interactions
Open this publication in new window or tab >>The crystal structure of a human PP2A phosphatase activator reveals a novel fold and highly conserved cleft implicated in protein-protein interactions
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2006 In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 281, 22434-22438 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25543 (URN)
Note
Part of urn:nbn:se:su:diva-8261Available from: 2008-10-23 Created: 2008-10-14Bibliographically approved
4. 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
5. 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

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