Strategizing for the purification of a multiple Big domain-containing protein in native conformation is worth it!

The reliability and accuracy of conformational or functional studies of any novel multidomain protein rely on the quality of protein. The bottleneck in structural studies with the complete Big_2 domain containing proteins like LigA, LigB or MpIBP is usually their large molecular size owing to their multidomain (>10-12 domains) architectures. Interestingly, a soil bacterium Paenarthrobacter aurescens TC1, harbours a gene that encodes a protein comprising of four predicted Big_2 domains. We report here the expression and purification of this novel, multiple Big_2 domains containing protein, Arig of P. aurescens TC1. During overexpression, recombinant Arig formed inclusion bodies and hence was purified by on-column refolding. The refolded Arig revealed a β-sheet conformation and a well-resolved near-UV CD spectra but did not exhibit a well-dispersed 2D [1 H-15 N]-HSQC NMR spectrum, as expected for a well-folded β-sheet native conformation. We, therefore, further optimized Arig overexpression in the soluble fraction by including osmolytes. CD spectroscopic and 2D [1 H-15 N]-HSQC analyses consolidate that Arig purified alternatively has a well-folded native conformation. While we describe different strategies for purification of Arig, we also present the spectral properties of this novel all-β-sheet protein.