Preparative purification process for human furin
View Patent ↗Recombinant truncated human furin was expressed in CHO cells and concentrated approximately 50-fold by ultrafiltration and diafiltration. The concentrate was purified by column chromatography on CAPTO MMC™ (mixed cation exchange/hydrophobic interaction gel) resulting in a 30-50 fold purification factor and a yield of at least 60%. The at least 20% pure preparation obtained after CAPTO MMC™ (mixed cation exchange/hydrophobic interaction gel) chromatography had already a purification degree allowing on-column maturation of pro-VWF. Then an additional Arginine Sepharose chromatography purification was carried out. This two column process for purification of truncated human furin resulted in an almost pure furin preparation with a specific activity of approximately 290,000 U furin/mg protein and a yield of about 50%.
1. A method of purifying a furin polypeptide or a furin polypeptide derivative having furin biological activity from a protein solution comprising the following steps:
(a) binding the furin polypeptide or furin polypeptide derivative to a mixed cation exchange/hydrophobic interaction resin that binds the furin polypeptide or furin polypeptide derivative at pH 6.0, and
(b) recovering the furin polypeptide or furin polypeptide derivative from the resin by elution.
2. The method of claim 1 , characterized in that said furin polypeptide derivative is a truncated furin polypeptide.
3. The method of claim 2 , characterized in that said truncated furin polypeptide is the furin polypeptide according to SEQ ID NO:1.
4. The method of claim 1 , wherein the elution is step-wise elution.
5. The method of claim 1 , wherein said furin polypeptide, or furin polypeptide derivative is bound to said mixed cation exchange/hydrophobic interaction resin in 10 mM sodium acetate, 1 mM CaCl 2 pH 6.0, washed with 30 mM sodium chloride, 10 mM sodium acetate, 1 mM CaCl 2 pH 6.0, and eluted with 230 mM sodium chloride, 10 mM sodium acetate, 1 mM CaCl 2 pH 6.0.
6. The method of claim 1 , wherein said furin polypeptide, or furin polypeptide derivative is bound to said mixed cation exchange/hydrophobic interaction resin in 50 mM Hepes, 1 mM CaCl 2 pH 6.0, washed with 30 mM sodium chloride, 50 mM Hepes, 1 mM CaCl 2 pH 6.0, and eluted with 230 mM sodium chloride, 50 mM Hepes, 1 mM CaCl 2 pH 6.0.
7. The method of claim 1 , wherein the elution is gradient elution.
8. The method of claim 7 , wherein said gradient is 0-500 mM NaCl in 10 mM sodium acetate, 1 mM CaCl 2 pH 6.0.
9. A method of recovering a furin polypeptide or a furin polypeptide derivative having furin biologic activity comprising the following steps:
(a) binding said furin polypeptide or furin polypeptide derivative to a mixed cation exchange/hydrophobic interaction resin,
(b) eluting said furin polypeptide or furin polypeptide derivative from the mixed cation exchange/hydrophobic interaction resin,
(c) binding the eluted furin polypeptide or furin polypeptide derivative to an Arginine-Sepharose resin, and
(d) eluting said furin polypeptide or furin polypeptide derivative from the Arginine-Sepharose resin.
10. The method of claim 9 , characterized in that said furin polypeptide derivative is a truncated furin polypeptide.
11. The method of claim 10 , characterized in that said truncated furin polypeptide has the sequence shown in SEQ ID NO:1.
12. The method of claim 2 , wherein said truncated furin polypeptide is bound to said mixed cation exchange/hydrophobic interaction resin in 10 mM sodium acetate, 1 mM CaCl 2 pH 6.0, washed with 30 mM sodium chloride, 10 mM sodium acetate, 1 mM CaCl 2 pH 6.0, and eluted with 230 mM sodium chloride, 10 mM sodium acetate, 1 mM CaCl 2 pH 6.0.
13. The method of claim 2 , wherein said truncated furin polypeptide is bound to said mixed cation exchange/hydrophobic interaction resin in 50 mM Hepes, 1 mM CaC12 pH 6.0, washed with 30 mM sodium chloride, 50 mM Hepes, 1 mM CaCl 2 pH 6.0, and eluted with 230 mM sodium chloride, 50 mM Hepes, 1 mM CaCl 2 pH 6.0.