Effector-assisted refolding of recombinant tissue-plasminogen activator produced in Escherichia coli.

Abstract:

: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.

authors

Grunfeld H,Patel A,Shatzman A,Nishikawa AH

doi

10.1007/BF02950781

subject

Has Abstract

pub_date

1992-05-01 00:00:00

pages

117-38

issue

2

eissn

0273-2289

issn

1559-0291

journal_volume

33

pub_type

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