title: Engineered solubility tag for solution NMR of proteins.
abstract: The low solubility of many proteins hinders large scale expression and purification as well as biophysical measurements. Here, we devised a general strategy to solubilize a protein by conjugating it at a solvent-exposed position to a 6 kDa protein that was re-engineered to be highly soluble. We applied this method to the CARD domain of Apoptosis-associated speck-like protein containing a CARD (ASC), which represents one member of a class of proteins that are notoriously prone to aggregation. Attachment of the tag to a cysteine residue, introduced by site-directed mutagenesis at its self-association interface, improved the solubility of the ASC CARD over 50-fold under physiological conditions. Although it is not possible to use nuclear magnetic resonance (NMR) to obtain a high quality 2D correlation spectrum of the wild type domain under physiological conditions, we demonstrate that NMR relaxation parameters of the solubilized variant are sufficiently improved to facilitate virtually any demanding measurement. The method shown here represents a straightforward approach for dramatically increasing protein solubility, enabled by ease of labeling as well as flexibility in tag placement with minimal perturbation to the target.
authors: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, 44106.
authors: Amy M
authors: AM
authors: Ruschak
authors: Justine D
authors: JD
authors: Rose
authors: Michael P
authors: MP
authors: Coughlin
authors: Tomasz L
authors: TL
authors: Religa
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism
chemical_list: D003598:Cytoskeletal Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D003545:Cysteine
keywords: CARD domain; nuclear magnetic resonance; protein A; protein aggregation; protein engineering; protein solubility
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