Clusterin purification from HEK293E cells

Express Clusterin (Clu) in HEK293E cells cultured in FreeStyle 293 Expression Medium (Thermo Fisher Scientific, 12338018) for 96h 0m 0s.

Centrifuge culture and keep conditioned medium.

Dialyze conditioned medium in 20millimolar (mM) Na acetate 5.0 (volume ratio <1:100).

If some precipitates are observed, remove by centrifugation.

Load dialyzed conditioned medium into a HiTrap SP XL cation exchange column previously equilibrated with 20millimolar (mM) Na acetate 5.0. Wash with 20millimolar (mM) Na acetate 5.0.

Wash the column with 10 column volumes (CV) of denaturing buffer (20millimolar (mM) Na acetate 5.0, 6Molarity (M) urea) to remove undesired proteins bound to Clu.

Wash the column with 5 CVs 20millimolar (mM) Na acetate 5.0.

Elute Clu with a 0millimolar (mM)-500millimolar (mM) linear NaCl gradient in 20millimolar (mM) Na acetate 5.0.

Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.

Note

Note: Denaturing wash can be omitted if not many contaminants are observed in the conditioned media. A small percentage of Clu in the conditioned media is not cleaved at the furin-like protease cleavage site (pre-secretory Clu), probably due to its high expression level. Clus is highly glycosylated and migrates in the denaturing SDS-PAGE gel at around 40 kDa (α and β chains, not resolved) or around 70 kDa (pre-secretory, uncleaved Clu)

Load Clu-containing fractions into a Superdex-200 previously equilibrated 20millimolar (mM) Na acetate 5.0, 100millimolar (mM) NaCl, 1millimolar (mM) EDTA.

Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.

Note

Clu oligomers are in equilibrium with monomeric Clu so all peaks containing Clu can be pooled. Oligomeric state of Clu is pH dependent. At pH 5.0 mainly monomeric Clu is eluted.

Concentrate Clu-containing fractions using a Vivaspin ultracentrifugation unit 10,000 MWCO or similar until reach desired concentration.

Aliquot and flash-freeze purified Clu in liquid nitrogen for storage at -80°C.

Note

Note: Approximate yield: from 250 ml of conditioned media around 12 mg of pure Clu are obtained. Clu purified from HEK293E cells present a comparable glycosylation pattern as Clu purified from plasma (Biovendor R&D, RD172034100). In order to obtain sharp bands of the α and β Clu chains in the SDS-PAGE, deglycosylation can be performed with PNGase F.