On-column refolding and purifying of lipoproteins
AssigneeMerck Sharp & Dohme Corp.
InventorsRebecca A. Chmielowski, Collette Cutler, Hong Li, Thomas O. Linden
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Abstract
Provided are methods of refolding and purifying lipoproteins on a chromatography column. In particular methods of refolding ApoL1 polypeptides using a hydrophobic interaction column (HIC). The present invention provides methods for the purification of an active form of a human lipoprotein, such as ApoL1. More particularly, the present invention relates to a method for renaturing an inclusion body of proteins expressed in a large quantity in E. coli into an active form using a hydrophobic interaction column and removal of impurities, e.g., endotoxins.
Claims19
A method of refolding and purifying a lipoprotein expressed in a bacterial cell comprising:
- a) solubilizing and unfolding inclusion bodies in a solubilization buffer;
- b) adjusting conductivity of the buffer containing the lipoprotein to at least 65-130 mS/cm;
- c) binding the lipoprotein on a hydrophobic interaction chromatography (HIC) column;
- d) washing the HIC column with a wash buffer;
- and
- e) eluting the lipoprotein with either a gradient elution buffer or a non-gradient elution buffer thereby yielding a HIC column eluent with a purity of at least 44%, as determined by reverse phase high performance liquid chromatography (RP-HPLC).
A method of removal of endotoxin for an apolipoprotein comprising:
- a) unfolding the apolipoprotein by addition of guanidine hydrochloride to an apolipoprotein containing pool collected from an anion exchange membrane;
- b) adding urea and ammonium sulfate to the pool;
- c) loading the apolipoprotein in a flowthrough mode on a hydrophobic interaction chromatography (HIC) column;
- d) washing the HIC column with an appropriate wash buffer;
- and
- d) collecting the flowthrough.
Dependencies are inferred from the claim text.