Abstract
The first described and best known mammalian secreted chaperone, abundant in human blood, is clusterin. Recent independent studies are now exploring the potential use of clusterin as a therapeutic in a variety of disease contexts. In the past, the extensive post-translational processing of clusterin, coupled with its potent binding to essentially any misfolded protein, have meant that its expression as a fully functional recombinant protein has been very difficult. We report here the first rapid and high-yield system for the expression and purification of fully post-translationally modified and chaperone-active clusterin. Only 5–6 days is required from initial transfection to harvest of the protein-free culture medium containing the recombinant product. Purification to near-homogeneity can then be accomplished in a single affinity purification step and the yield for wild type human clusterin is of the order of 30–40 mg per litre of culture. We have also shown that this system can be used to quickly express and purify custom-designed clusterin mutants. These advances dramatically increase the feasibility of detailed structure–function analysis of the clusterin molecule and will facilitate identification of those specific regions responsible for the interactions of clusterin with receptors and other molecules.
Highlights
The first described and best known mammalian secreted chaperone, abundant in human blood, is clusterin
To further optimise culture conditions for the expression of CLU, MEXi293E cells were transfected with expression plasmids encoding wild type human CLU (WT CLU) incorporating a twin Streptag at the C-terminus of the α-chain or the C-tag at the C-terminus of the β-chain
Several important lessons taken from previous attempts to generate recombinant CLU can be summarised as follows
Summary
The first described and best known mammalian secreted chaperone, abundant in human blood, is clusterin. Clusterin (CLU) is the first reported abundant extracellular mammalian chaperone and promiscuously interacts with misfolded proteins to stabilise them in a soluble form[1,2,3] This activity is similar to that of the small heat shock proteins and places CLU in the class of chaperones known as “holdases”[3]. The protein is heavily N-glycosylated at six sites, to give 17–27% carbohydrate by mass; the protein has an apparent mass of 75–80 kDa in SDS PAGE, the actual mass is ~ 58–63 kDa12 This complex post-translational processing together with its propensity to form heterogenous oligomers in solution[1] have frustrated all previous attempts to determine its three-dimensional structure. A large part of the explanation for this lack of progress in the 35 years since CLU was discovered has been the lack of a tractable platform to manipulate its structure by mutations and efficiently express/purify the wild type protein and mutants for study
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