Production and purification of TAL6003: A novel version of the protease plasmin
| Autor: | Charles Miller, Myles Lindsay, Thomas P. Zimmerman, James Rebbeor, Rebecca Silverstein, Jennifer A. Hunt, Kenya Stokes, Anthony Caronna, Edward Koepf, Remil Aguda |
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| Rok vydání: | 2013 |
| Předmět: |
Protein Folding
Plasmin medicine.medical_treatment Gene Expression Affinity chromatography Benzamidine Sepharose chemistry.chemical_compound Kringles Zymogen medicine TAL6003 (recombinant plasmin) Escherichia coli Humans Amino Acid Sequence Fibrinolysin Cloning Molecular Serine protease Zymogen activation Inclusion Bodies Protease Chromatography biology Lysine chemistry Biochemistry biology.protein Oxidative refolding medicine.drug Peptide Hydrolases Biotechnology |
| Zdroj: | Protein Expression and Purification. 88(1):41-46 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2012.11.013 |
| Popis: | TAL6003 is an engineered form of human plasmin under development for thrombolytic therapy. TAL6003 (38.2kDa) contains nine disulfide bonds distributed within and between its two functional domains, a 25.2kDa serine protease domain linked to a 13.0kDa kringle I domain; kringles 2–5 present in native human plasmin have been deleted from this plasmin molecule. TAL6003 is expressed as its zymogen in Escherichia coli and is harvested in inclusion bodies. Following solubilization of inclusion bodies with urea as the chaotrope and glutathione as the reducing agent, this zymogen is refolded by dilution to a final concentration of 0.5mg/ml, with a yield of 48% (relative to total zymogen). Refolded TAL6003 zymogen is filtered, diafiltered, and filtered again prior to capture and purification on SP Sepharose, which is highly effective in removing host-cell protein. Subsequent affinity purification on ECH-Lysine Sepharose serves to capture polypeptide chains containing correctly refolded kringle 1 domain, which is the locus of the molecule’s lysine-binding site, and to further eliminate host-cell protein. TAL6003 zymogen eluted from the ECH-Lysine Sepharose column is activated to TAL6003 with streptokinase, with an activity yield of approximately 80%. Proteolytically active TAL6003 is stripped of streptokinase by passage through an anion-exchange (Q) membrane and is then affinity purified on Benzamidine Sepharose, which serves to remove unreacted TAL6003 zymogen and proteolytically degraded TAL6003. An ultrafiltration/diafiltration step, to concentrate and to formulate TAL6003, completes the purification process. The final product exhibited a specific activity of close to unity and high purity by several relevant criteria. |
| Databáze: | OpenAIRE |
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