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Method for producing recombinaton urokinase

A technology of prourokinase and urea, applied in the field of production of recombinant urokinase, which can solve problems such as safety and reproducibility concerns

Inactive Publication Date: 2004-10-20
PROTEOMTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Urokinase for clinical use is purified from collected urine, with safety and reproducibility concerns

Method used

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  • Method for producing recombinaton urokinase
  • Method for producing recombinaton urokinase
  • Method for producing recombinaton urokinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Refolding and purification of recombinant prourokinase

[0062] The kidney cDNA library was amplified by PCR to generate a DNA fragment encoding human prourokinase. The primers used in the above PCR reaction were UK-1 (5'-CATATGTCCAACGAACTGCACCAGGTTCCATCGAACTGTGACTGTC-3'[SEQ ID NO: 3]) and UK-2(5'- CTCGAGTTAGAGGGCCAGGCCATTCTCTTC-3' [SEQ ID NO: 4]). Primer UK-1 was used to introduce 6 silent mutations into the prourokinase gene, which can increase the expression efficiency in E. coli.

[0063] The full-length PCR product was cloned into pCR2.1 TOPO (Invitrogen) and sequenced from both ends using M13F and M13R primers. Figure 1 shows the nucleotide and its encoded protein sequence. The insert was excised using NdeI-XhoI restriction digestion, gel purified, and cloned into NdeI-XhoI digested pET43 (Novagen).

[0064] The pro-urokinase expression vector was transformed into Escherichia coli BL21 (DE3) strain and spread on the ZB plate containing ampicillin. ...

Embodiment 2

[0072] Example 2: Purification of recombinant prourokinase by heparin affinity chromatography and hydroxyapatite chromatography

[0073] The refolded pro-urokinase produced as described in Example 1 was concentrated by ultrafiltration (MilliporePellicon, 10,000Da cut-off membrane), and then subjected to SEC. A SEPHACRYL® S-300 column equilibrated with 1 mM EDTA, pH 8.0. Refolded prourokinase is separated from misfolded high molecular weight aggregates by this chromatography.

[0074] The urokinase-containing fraction was collected from the S-300 column, diluted 5-fold with 25 mM HEPES pH 7.0, and loaded into HiTrap TM Heparin HP agarose affinity chromatography column (Pharmacia / Amersham). The heparin affinity chromatography column was first equilibrated with 25mM HEPES pH 7.0, 25mM NaCl. After the sample is loaded on the column, wash the column with equilibration buffer until no protein is detected in the effluent. Thereafter, the chromatographic column was washed with ...

Embodiment 3

[0075] Example 3: Identification of recombinant urokinase

[0076] The purified human prourokinase produced according to Example 1 was diluted to 1 μM with 50 mM tris, 50 mM NaCl, 0.01% TWEEN® 20, pH 8.9, and incubated with plasmin (0.1 μg / mL) at 37° C. 60 minutes to convert to urokinase. The reaction was terminated by adding excess (12,500 IU / mL) aprotinin.

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Abstract

Highly efficient methods of producing properly folded recombinant urokinase are provided. Denatured recombinant pro-urokinase is refolded by first solubilizing the protein with a chaotroph at high pH, followed by refolding in the presence of reduced concentrations of chaotroph while the pH is slowly reduced.

Description

[0001] Cross references to related applications [0002] This application claims priority to US Provisional Patent Application 60 / 463,632 (filed April 16, 2003) and US Provisional Patent Application 60 / 498,134 (filed August 26, 2003), both of which are incorporated herein by reference in their entirety. Background of the invention [0003] Urokinase is a serine protease that cleaves plasminogen to generate active plasmin and thus plays an important role in fibrinolysis. Clinically, this activity has been used for the active treatment of thrombosis, including thrombotic stroke, pulmonary embolism, deep vein thrombosis, and others. [0004] Urokinase is initially synthesized as a 411 amino acid precursor protein with 12 intrachain disulfide bonds. Prourokinase is activated to mature urokinase by proteolysis in which the backbone is at Lys 158 Afterwards, it breaks to form a double-stranded molecule linked by disulfide bonds. Urokinase for clinical use is purified from collect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48C07K1/14C12N9/72
CPCC12N9/6462C12Y304/21073
Inventor 蔺新力
Owner PROTEOMTECH
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