Overexpression of aprE2, a Fibrinolytic Enzyme Gene from Bacillus subtilis CH3-5, in Escherichia coli and the Properties of AprE2

초록

The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at 20 degrees C, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at 37 degrees C. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of 1,069.4 +/- 42.4 U/mg protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and 40 C, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded A alpha and B beta chains of fibrinogen quickly, but not the gamma-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The K-m and k(cat)/K-m of AprE2 was 0.56 mM and 3.10 x 10(4) S-1M-1, respectively.

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