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 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.
Thakur
Krishan Gopal
KG
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Jaiswal
Ravi Kumar
RK
Shukla
Jinal K
JK
Praveena
T
T
Gopal
B
B
D001426:Bacterial Proteins / Q000302:isolation & purification*; D001671:Biochemistry / Q000379:methods*; D046912:Multiprotein Complexes / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D012808:Sigma Factor / Q000302:isolation & purification*
D001426:Bacterial Proteins; D046912:Multiprotein Complexes; D012808:Sigma Factor