Biochemical Characterization and In Vivo Testing of a Recombinant Fibrinolytic Enzyme from Bacillus sp. N18
Jing Huang 1, Wei-Jun Fang 1, Xin-Xia Peng 1, Lin-Fa Wang 1,2 , and Zi-Rong Wu 1*
1Molecular Biology Laboratory, School of Life Science, East China Normal University, Shanghai 200062, China
2CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia
Abstract.
Bacillus sp. N18, which produces a strongly fibrinolytic enzyme, was isolated from soil samples. The gene coding for the fibrinolytic enzyme (N18) was cloned and high level of expression/secretion was achieved using a high copy number plasmid and a triple protease deficient Bacillus subtilis strain. The recombinant enzyme (termed ReN18) was purified by consecutive chromatography with Streamline SP XL and Sephacryl S-100, resulting in a single protein band on IEF gel with an isoelectric point approximately at pH 8.6 and on SDS-PAGE with an apparent molecular weight of 28,000 Da, which is very close to the molecular mass determined by mass spectrometry (27,728 Da). N-terminal sequencing revealed that the first 15 amino acid residues are AQSVPYGISQIKAPA , identical to those deduced from DNA sequence. The purified ReN18 showed a higher affinity for fibrin in comparison with urokinase or plasmin in vitro. ReN18 was also active in vivo in experimental animals using either oral or intravenous route of administration. Furthermore, analysis of plasmin (Plm) activity, plasminogen activator inhibitor (PAI) activity and D-dimer concentration, and residual plasma fibrinogen (Fbg) indicated that the recombinant enzyme was able to cleave directly cross-linked fibrin without concurrently destroying fibrinogen. Results obtained from a brain-thrombus mouse model demonstrated that the recombinant enzyme was able to increase the surviving rate of experimental animals to 82% at a dose of 8,000 U/kg, 77% at 4,000 U/kg, 40% at 2,000 U/kg, respectively, in comparison with 57% at a dose of 8,000 U/kg when urokinase was used. The current study demonstrated that ReN18 could be used as a potent thrombolytic agent.
Key words: Bacillus, Fibrinolytic enzyme, Nattokinase, Plasmin, Thrombolytic agent, Urokinase
*Author for Correspondence.
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