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,Patel A,Shatzman A,Nishikawa AH




Has Abstract


1992-05-01 00:00:00












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    authors: Costa AC,Dechechi EC,Silva FL,Maugeri F,Maciel R

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  • A novel phosphoramidite method for automated synthesis of oligonucleotides on glass supports for biosensor development.

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    authors: Sojka B,Piunno PAE,Wust CC,Krull UJ

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  • Depletion of human lymphocytes from peripheral blood and bone marrow by affinity ligands conjugated to agarose-polyacrolein microsphere beads.

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    journal_title:Applied biochemistry and biotechnology

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    authors: Weiss L,Margel S,Slavin S

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  • Development of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of ACT Producing Alternaria alternata, the Agent of Brown Spot Disease in Tangerine.

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    authors: Moghimi H,Moradi A,Hamedi J,Basiri M

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    authors: Sivaramakrishnan R,Muthukumar K

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    authors: Ennis MP,Wisdom GB

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    authors: Chen H,Qin L,Li H

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    authors: Mishra V,Jana AK

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    authors: Bruins ME,Janssen AE,Boom RM

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    authors: Tang B,Bi W,Row KH

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  • A Comprehensive Study on Chlorella pyrenoidosa for Phenol Degradation and its Potential Applicability as Biodiesel Feedstock and Animal Feed.

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    journal_title:Applied biochemistry and biotechnology

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    authors: Tanaka H,Ebata T,Kuwahara I,Matsuo M,Ogbonna JC

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    authors: Yin C,Zheng L,Chen L,Tan Q,Shang X,Ma A

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    authors: Converse AO

    更新日期:2007-04-01 00:00:00

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    authors: Kirkkopru I,Alpaslan C,Omay D,Güvenilir Y

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    authors: Bhattacharya S,Schiavone M,Nayak A,Bhattacharya SK

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    journal_title:Applied biochemistry and biotechnology

    pub_type: 杂志文章


    authors: Xu P,Yano T,Yamamoto K,Suzuki H,Kumagai H

    更新日期:1996-03-01 00:00:00