Methods for purifying highly anionic proteins
View Patent ↗The present invention provides a process for isolating and purifying highly anionic target proteins, for example, sulfated proteins. Sulfated proteins have five (5) or more, sulfations. In a preferred embodiment, the sulfated protein has six (6) sulfations, e.g., six sulfations on N-terminal tyrosine residues, as embodied in PSGL-1 (P-selectin glycoprotein ligand).
1. A method for purifying a protein in a sample from a plurality of DNA/histone complexes, comprising a step of loading the sample containing the protein on a metal chelate chromatography substrate wherein the protein is captured on the substrate, and a step of washing the substrate, wherein at least one of the loading step and the washing step uses a solution comprising at least about 2M NaCl to remove DNA from the sample, thereby purifying the protein in the sample.
2. The method of claim 1 , wherein the protein is highly anionic.
3. The method of claim 2 , wherein the protein is hypersulfated.
4. The method of claim 3 , wherein the protein is PSGL-1.
5. The method of claim 1 , wherein the loading step uses the solution comprising at least about 2M NaCl to remove DNA from the sample.
6. The method of claim 1 , wherein the washing step uses the solution comprising at least about 2M NaCl to remove DNA from the sample.
7. A method for purifying a protein in a sample from a plurality of DNA/histone complexes, comprising a step of loading the sample containing the protein on a metal chelate chromatography substrate wherein the protein is captured on the substrate, and a step of washing the substrate, wherein at least one of the loading step and the washing step uses a solution comprising an ionic strength of at least about 2M to remove DNA from the sample, thereby purifying the protein in the sample.
8. The method of claim 7 , wherein the protein is highly anionic.
9. The method of claim 8 , wherein the protein is hypersulfated.
10. The method of claim 9 , wherein the protein is PSGL-1.
11. A method for purifying a highly anionic protein in a sample from a plurality of DNA/histone complexes, comprising a step of loading the sample containing the protein on a hydrophobic interaction chromatography substrate wherein the protein is captured on the substrate, and a step of washing the substrate, wherein the washing step uses a solution comprising either at least about 5% ethanol or at least about 5% isopropanol to remove DNA from the sample, thereby purifying the protein in the sample.
12. The method of claim 11 , wherein the protein is hypersulfated.
13. The method of claim 12 , wherein the protein is PSGL-1.