Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8359512 | Protein Expression and Purification | 2018 | 7 Pages |
Abstract
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 [1H-15N]-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 [1H-15N]-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.
Keywords
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Asmita D. Pawar, Deepshikha Verma, Venu Sankeshi, Rajeev Raman, Yogendra Sharma,