Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Preparation of High-Purity Urokinase Using Single-Step Hydrophobic Interaction Chromatography with p-Aminobenzamidine Ligand

  • Cao, Xue-Jun (State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science and Technology) ;
  • Zhou, Jian-Hua (Research Center, Shanghai No.1 Biochemical and Pharmaceutical Company) ;
  • Huang, Zhen-Hui (Research Center, Shanghai No.1 Biochemical and Pharmaceutical Company) ;
  • Wu, Xing-Yan (State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science and Technology) ;
  • Hur, Byung-Ki (Department of Biological Engineering, Inha University)
  • Published : 2002.04.01
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Abstract

A novel process for urokinase purification was studied using p-aminobenzamidine as the ligand and sepharose 4B as the matrix. The adsorption, washing, and elution conditions were optimized by an unusual method. An adsorption buffer containing 2.5 M NaCl and $1\%$ Tween 80 facilitated the adsorption of urokinase on the affinity media and prevented contaminants from binding to the p-aminobenzamidine affinity gel. It was found that $5\%$ Tween 80 removed most of the contaminants from the affinity column. A 0.2 M glycine elution buffer containing 0.5 M NaCl (pH 3.0) was found to have a strong elution ability with a high recovery and purity of urokinase. A crude urokinase material of231 IU/mg protein from human urine was purified to 124,300 IU/mg protein with a purification factor of 538 and yield of $86.7\%$. As a result, a high purity urokinase was obtained with only a single affinity chromatography step. The purification process was successfully scaled-up to a 2-1 chromatography column. The resulting urokinase eluate could be directly lyophilized, thereby complying with Chinese pharmacopoeia (1995 version) standards.

Keywords

References

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