title: Expression, purification, and characterization of human cystatin C monomers and oligomers.
abstract: Human cystatin C (cysC) is a soluble basic protein belonging to the cysteine protease inhibitor family. CysC is a potent inhibitor of cathepsins--proteolytic enzymes that degrade intracellular and endocytosed proteins, remodel extracellular matrix, and trigger apoptosis. Inhibition is via tight reversible binding involving the N-terminus as well as two β-hairpin loops of cysC. As a significant component of cerebrospinal fluid, cysC has numerous other functions, including support of neural stem cell growth and differentiation. Several studies suggest that cysC may bind to the Alzheimer-related protein beta-amyloid (Aβ), and inhibit its aggregation and toxicity. Because of an increasing recognition of its important biological roles, there is considerable interest in methods to produce full-length recombinant human cysC. Several researchers have reported success, but with processes that require multiple purification steps. Here we report successful production of human cysC using an intein-based expression system and a simple one-column purification scheme. The recombinant protein so obtained was natively folded and active as an enzyme inhibitor. Unexpectedly, even mild concentration by ultrafiltration caused significant oligomerization. The oligomers are noncovalent and retain the native secondary structure and inhibitory activity of the monomer. The oligomers, but not the monomers, were highly effective at inhibiting aggregation of Aβ. These results demonstrate the critical importance of careful physicochemical characterization of recombinant cysC protein prior to evaluation of its biological functions.
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, United States.
authors.forename: Tyler J
authors.initials: TJ
authors.lastname: Perlenfein
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, United States. Electronic address: regina.murphy@wisc.edu.
authors.forename: Regina M
authors.initials: RM
authors.lastname: Murphy
mesh_terms: D055316:Cystatin C / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D017510:Protein Folding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C525030:CST3 protein, human; D055316:Cystatin C; D011994:Recombinant Proteins
keywords: Aggregation; Amyloid; Beta-amyloid; Cystatin C; Light scattering; Oligomers