Granted Patent B2

US 9,206,410 · App. 13/147,491 · Granted Dec 8, 2015

Abstract

Claims (10)

1. A method for preparing a recombinant plasminogen having a single kringle domain, a peptide linker sequence, an activation site, and a serine protease domain, wherein said single kringle domain is at least 90% identical to kringle 1 or kringle 4 of SEQ ID NO: 2; said peptide linker sequence between said single kringle domain and said serine protease domain consists of residues 91-94 of SEQ ID NO: 1; and said method comprises: contacting a composition comprising said recombinant plasminogen with a cation-exchange medium under a cation-exchange condition that is sufficient for said cation-exchange medium to bind said recombinant plasminogen; wherein said cation-exchange medium comprises a sulfopropyl ligand coupled to an agarose support.

2. The method of claim 1 further comprising: eluting the recombinant plasminogen bound by the cation-exchange medium to obtain a cation-exchange medium eluate comprising the recombinant plasminogen.

3. The method of claim 2 further comprising: contacting the cation-exchange medium eluate with a first affinity medium under a first affinity condition that is sufficient for the affinity medium to bind the recombinant plasminogen.

4. The method of claim 3 , wherein the first affinity medium comprises a ligand coupled to a support, wherein the ligand has affinity for the recombinant plasminogen.

5. The method of claim 4 , wherein the ligand is lysine, wherein the support is an agarose.

6. The method of claim 1 further comprising: expressing the recombinant plasminogen using a recombinant expression system.

7. The method of claim 6 , wherein the step of expressing comprises performing the expression system under an expression condition sufficient to produce a recombinant plasminogen inclusion body.

8. The method of claim 7 further comprising: contacting the recombinant plasminogen inclusion body with a solubilization buffer under a solubilization condition sufficient to obtain a solubilized recombinant plasminogen inclusion body.

9. The method of claim 8 further comprising: contacting the solubilized recombinant plasminogen inclusion body with a refolding solution under a refolding condition to obtain a composition comprising the recombinant plasminogen.

10. The method of claim 1 further comprising: adding a polyethylene glycol (PEG) or a salt to a refolding solution under a precipitation condition, wherein the refolding solution comprises the recombinant plasminogen and aggregated polypeptides, wherein the recombinant plasminogen is refolded recombinant plasminogen, wherein the precipitation condition is sufficient to precipitate all or a substantial portion of the aggregated proteins.

Assignments (24)