title: Production and purification of TAL6003: a novel version of the protease plasmin.
abstract: 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 25.2 kDa serine protease domain linked to a 13.0 kDa 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.
authors.affiliation: Process Development and Technology Department, Grifols Therapeutics Inc., 1200 New Hope Road, Raleigh, NC 27610, United States.
authors.forename: Remil
authors.initials: R
authors.lastname: Aguda
authors.forename: Anthony
authors.initials: A
authors.lastname: Caronna
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hunt
authors.forename: Edward
authors.initials: E
authors.lastname: Koepf
authors.forename: Myles
authors.initials: M
authors.lastname: Lindsay
authors.forename: Charles
authors.initials: C
authors.lastname: Miller
authors.forename: James
authors.initials: J
authors.lastname: Rebbeor
authors.forename: Rebecca
authors.initials: R
authors.lastname: Silverstein
authors.forename: Kenya
authors.initials: K
authors.lastname: Stokes
authors.forename: Thomas
authors.initials: T
authors.lastname: Zimmerman
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005341:Fibrinolysin / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D018082:Kringles / Q000235:genetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D017510:Protein Folding
chemical_list: D010447:Peptide Hydrolases; D005341:Fibrinolysin; D008239:Lysine