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Renaturation separation purification method of tissue plasminogen activator mutant

A technology of plasminogen and activator, which is applied in the field of separation and purification of recombinant protein in medical biotechnology, can solve problems such as high cost, and achieve the effects of reducing loss, saving time and reducing production cost.

Inactive Publication Date: 2004-12-22
CHINA PHARM UNIV
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  • Abstract
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Problems solved by technology

However, refolding reteplase with reduced and oxidized glutathione and other refolding reagents in a certain ratio can be successful, but the cost is very expensive.

Method used

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  • Renaturation separation purification method of tissue plasminogen activator mutant

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Embodiment Construction

[0011] The preparation method of reteplase is as follows: the expressed reteplase engineered bacterial cells are collected, suspended in 20 mM Tris-Hcl pH8.0, ultrasonically broken and centrifuged at 12000 rpm at 4° C. for 15 minutes to collect inclusion bodies. Inclusion body solution I, its components are 6~8M urea, 10~100mM Tris-Hcl, 0.5M NaCl, 1~100mM imidazole, 0~5mM 2-mercaptoethanol pH8.0, stirred at room temperature, fully dissolved, 4 Centrifuge at 12000 rpm for 15 minutes, take the supernatant, and filter it with a 0.22um or 0.45um filter membrane to obtain a denatured reteplase solution.

[0012] The denatured reteplase was passed through the Ni solution equilibrated with solution I before 2+ -HiTrap column, after loading the sample, first use twice the column volume of solution I and solution II, the components of solution II are 6~8M urea, 10~100mM Tris-Hcl, 0.5M NaCl, 20~500mM imidazole, 0~ 100mM 2-mercaptoethanol PH7~10, wash once each, and then use six times c...

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Abstract

A process for renaturating, separating and purifying the mutant of tissue plasminogen activator features that the imidazole and urea are used to make the tissue plasminogen activator 'Ruitipu enzyme' be correctly folded in order to have activity and make it be renaturated, separated and purified simultaneously.

Description

1. Technical field [0001] The invention belongs to the technical field of separation and purification of recombinant protein of medical biotechnology. 2. Background technology [0002] Reteplase is a gene recombinant human tissue plasminogen activator mutant expressed in Escherichia coli by site-directed mutagenesis technology. It is a single-chain aglycosylated polypeptide, containing 355 amino acids, and has the 1-3, 176-527 amino acid coding sequence of natural human tissue plasminogen activator (tissue plasminogen activator, t-PA). Reteplase, which only carries the kringle2 domain and protease domain of wild-type t-PA, has become a comparative therapy because of its long half-life of 16-20 minutes, faster and more thorough thrombolysis and less risk of intracranial hemorrhage. Ideal thrombolytic drug. [0003] Since reteplase contains 10 pairs of disulfide bonds, it forms inclusion bodies when expressed in Escherichia coli, and it is difficult to correctly fold and ref...

Claims

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Application Information

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IPC IPC(8): C07K1/14C12N9/00
Inventor 沈子龙廖建民孙石静张新元
Owner CHINA PHARM UNIV
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