Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis σ/anti-σ factor protein complexes

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determin...

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Bibliographic Details
Published in:Protein expression and purification 2010-12, Vol.74 (2), p.223-230
Main Authors: Thakur, Krishan Gopal, Jaiswal, Ravi Kumar, Shukla, Jinal K., Praveena, T., Gopal, B.
Format: Article
Language:English
Subjects:
Anti-sigma factor
Bacterial Proteins - isolation & purification
Biochemistry - methods
Co-expression
Multiprotein Complexes - isolation & purification
Mycobacterium tuberculosis
Mycobacterium tuberculosis - chemistry
Protein complex
Recombinant
Sigma factor
Sigma Factor - isolation & purification
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Summary:The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the σ factor/anti-σ factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of σ factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2010.06.018