Effector-assisted refolding of recombinant tissue-plasminogen activator produced in Escherichia coli.
Recombinant tissue-plasminogen activator (r-tPA), expressed in Escherichia coli cells in an aggregated form, was solubilized with a strong chaotrope in the absence of any reducing agent. The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein. The use of partially purified fibrinogen, as a source of fibrin (the effector), is shown to facilitate the reactivation process and increase its yield by at least a factor of two. The yield of the process is also shown to be particularly dependent on the recombinant protein concentration. At a concentration level of 3-3.7 mg r-tPA/L in the reactivation mixture, up to a 90% yield of activity was obtained. Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme. This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column. The product was composed of roughly equal amounts of one-chain and two-chain t-PA. The feasibility of using a two water-soluble polymeric phase system, with a centrifugal partition chromatography (CPC), in scaling up the reactivation process or the purification step was also evaluated.
Grunfeld
H
H
Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Patel
A
A
Shatzman
A
A
Nishikawa
A H
AH
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003911:Dextrans; D004926:Escherichia coli / Q000378:metabolism*; D005337:Fibrin / Q000494:pharmacology; D005340:Fibrinogen / Q000494:pharmacology; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D011092:Polyethylene Glycols; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D003911:Dextrans; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D005337:Fibrin; D005340:Fibrinogen; D010959:Tissue Plasminogen Activator; D005978:Glutathione