Method for purifying a protein of the cystine-knot superfamily
AssigneeBoard of Regents of the University of Nebraska by and on Behalf of the University of Nebraska Medical Center
InventorsElliott Bedows, Jason A. Wilken
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Abstract
The present invention relates to a two-step method for isolating proteins from the cystine-knot superfamily based on dye ligand affinity chromatography and reversed-phase chromatography. Advantageously, the method can be performed in a relatively short period of time, involves inexpensive reagents, and requires little sample preparation before and during the purification process. Protein fusions between cystine-knot proteins and proteins of interest are further provided for the isolation of said protein of interest or complexes containing said protein of interest using the two-step method disclosed.
Claims19
A method for purifying a protein of the cystine-knot superfamily comprising:
- (a) applying a sample containing a protein of the cystine-knot superfamily to a dye affinity chromatography matrix comprising a dye ligand, wherein the sample is selected from the group consisting of a tissue, a bodily fluid and a culture medium;
- (b) removing contaminants from the dye affinity chromatography matrix;
- (c) eluting the cystine-knot protein from the dye affinity chromatography matrix;
- (d) applying the eluted cystine-knot protein to a reversed-phase chromatography matrix;
- (e) removing contaminants from the reversed-phase chromatography matrix;
- and (f) eluting the cystine-knot protein from the reversed-phase chromatography matrix thereby purifying the protein of the cystine-knot superfamily.
A method for purifying a select protein of interest comprising:
- (a) obtaining a first sample containing a fusion protein comprising a protein of the cystine-knot superfamily, or fragment thereof, which comprises requisite cysteines necessary to fold into the cystine-knot conformation and binds to an affinity dye ligand, operably linked with a select protein of interest by a protease cleavage site;
- (b) applying said first sample to a dye affinity chromatography matrix comprising a dye ligand so that the cystine-knot protein, or fragment thereof, of the fusion protein binds to the dye affinity chromatography matrix;
- (c) removing contaminants from the dye affinity chromatography matrix;
- and
- (d) eluting the select protein of interest from the dye affinity chromatography matrix by applying a protease which specifically cleaves the protease cleavage site thereby substantially purifying the select protein of interest.
Dependencies are inferred from the claim text.