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.

Insights into the mechanism of cystatin C oligomer and amyloid formation and its interaction with β-amyloid

“…In previous work, we developed a simplified affinity chromatography-based protocol for production of recombinant CysC (37). During the routine protein concentration, we discovered CysC contained some oligomers.…”

“…Recently, we reported on the production and purification of recombinant human CysC. We discovered that a fraction of the protein readily associates into small soluble oligomers (37), raising the question as to whether these oligomers are intermediates to amyloid fibrils. In this report, we test the proposal that CysC forms fibrils by propagated domain-swapping, we investigate the structure of oligomeric CysC, and we probe the role of oligomers in the CysC amyloidogenesis pathway.…”

Effect of channel-induced shear on biologics during ultrafiltration/diafiltration (UF/DF)

“…6, and confirmed at least qualitatively, using Vmax. As detailed by Perlenfein and Murphy [13], the scattering intensity is a linear function of mass concentration and a sixth order function of particle size. Thus, a broader distribution on a logarithmic plot demonstrates a significant increase in particle size.…”

“…Moreover, concentration dependent effects become important during ultrafiltration especially on recirculation at higher protein concentrations, where concentration dependent aggregation becomes important. Perlenfein and Murphy [13] reported on the aggregation of human cystatin C as a function of concentration during centrifugal ultrafiltration. From past phenomenological observations, it could be concluded that increased solid-liquid interface contact in tight to medium screen channels, higher wall shear stress during the operation, and concentration dependent effects together may act to produce aggregates and particulates.…”

“…In addition to the above mentioned stresses that processing may cause, UF/DF is a very dynamic unit operation where the protein concentration increases with volume reduction and a change in buffer matrix occurs during diafiltration. The general propensity of several proteins to aggregate with concentration [9,13,14] and the processing-related stresses indicate that it is important to understand and control the process in such a way that impact on product quality is minimal.…”