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

Abstract

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.

Selectivity among Anti-σ Factors by Mycobacterium tuberculosis ClpX Influences Intracellular Levels of Extracytoplasmic Function σ Factors

“…As the purified anti-factors are prone to aggregation, the purified substrates used in these experiments consisted of the ECF factor complexed with an anti-factor containing the degron at the C terminus. Coexpression and copurification of the /antifactor complexes significantly improves the yield of homogenous protein samples, as the or anti-factors in isolation are relatively unstable (29). The segment comprising the last three residues (9-11) of the ssrA degron was shown to interact with E. coli ClpX (19).…”

An extracytoplasmic function sigma factor cotranscribed with its cognate anti-sigma factor confers tolerance to NaCl, ethanol and methylene blue in Azospirillum brasilense Sp7

“…In order to avoid the problems resulting from the unequal solubility and stability of the two interacting partner proteins when overexpressed separately (Thakur et al, 2010), we examined the physical interaction between RpoE and ChrR by cloning both genes (rpoE-chrR) together in pET15b to coexpress (i) His-RpoE from the Shine-Dalgarno (SD) sequence of the vector and (ii) wild-type ChrR from its native SD sequence located at the 39 end of the rpoE. We observed that the amount of the two overexpressed and eluted proteins was unequal (Fig.…”

“…4b, ii and iii). Another Role of RpoE in A. brasilense co-expression study of sigma factors and anti-sigma factors has shown that the expression of the promoter distal gene is usually lower than the promoter proximal gene, resulting in a substoichiometric complex of the two co-expressed proteins (Thakur et al, 2010). Since only His-RpoE can directly bind to the Ni-NTA agarose, presence of ChrR along with His-RpoE in the eluted fraction indicates that ChrR formed a complex with RpoE (Fig.…”

Mycobacterium tuberculosis RsdA provides a conformational rationale for selective regulation of σ-factor activity by proteolysis

“…RsdA is the cognate anti-σ factor for σ D and full-length σ D interacts with the cytosolic component of RsdA (30). Crystallization trials were performed to obtain crystals of full-length σ D and the ASD of RsdA (RsdA 1 − 80 ).…”