Target Selection of Soluble Protein Complexes for Structural Proteomics Studies

Resource type
Date created
2005
Authors/Contributors
Author: Yun, Steven
Author: Tam, Bonny
Author: Dalal, Kush
Abstract
Background: Protein expression in E. coli is the most commonly used system to produce proteinfor structural studies, because it is fast and inexpensive and can produce large quantity of proteins.However, when proteins from other species such as mammalian are produced in this system,problems of protein expression and solubility arise [1]. Structural genomics project are currentlyinvestigating proteomics pipelines that would produce sufficient quantities of recombinant proteinsfor structural studies of protein complexes. To investigate how the E. coli protein expressionsystem could be used for this purpose, we purified apoptotic binary protein complexes formedbetween members of the Caspase Associated Recruitment Domain (CARD) family.Results: A combinatorial approach to the generation of protein complexes was performedbetween members of the CARD domain protein family that have the ability to form hetero-dimersbetween each other. In our method, each gene coding for a specific protein partner is cloned inpET-28b (Novagen) and PGEX2T (Amersham) expression vectors. All combinations of proteincomplexes are then obtained by reconstituting complexes from purified components in nativeconditions, after denaturation-renaturation or co-expression. Our study applied to 14 solubleCARD domain proteins revealed that co-expression studies perform better than native anddenaturation-renaturation methods. In this study, we confirm existing interactions obtained invivoin mammalian cells and also predict new interactions.Conclusion: The simplicity of this screening method could be easily scaled up to identify solubleprotein complexes for structural genomic projects. This study reports informative statistics on thesolubility of human protein complexes expressed in E.coli belonging to the human CARD proteinfamily.
Document
Published as
Proteome Science 2005, 3:3 doi:10.1186/1477-5956-3-3
Publication title
Proteome Science
Document title
Target Selection of Soluble Protein Complexes for Structural Proteomics Studies
Date
2005
Volume
3
Issue
3
Publisher DOI
10.1186/1477-5956-3-3
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Copyright is held by the author(s).
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Peer reviewed?
Yes
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Member of collection
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