title: Overexpression of aprE2, a fibrinolytic enzyme gene from Bacillus subtilis CH3-5, in Escherichia coli and the properties of AprE2.
abstract: 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°C, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at 37°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 Aα and Bβ chains of fibrinogen quickly, but not the γ-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The Km and kcat/Km of AprE2 was 0.56 mM and 3.10 × 10(4) S(-1) M(-1), respectively.
authors.affiliation: Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors.forename: Seon-Ju
authors.initials: SJ
authors.lastname: Jeong
authors.forename: Kye Man
authors.initials: KM
authors.lastname: Cho
authors.forename: Chang Kwon
authors.initials: CK
authors.lastname: Lee
authors.forename: Gyoung Min
authors.initials: GM
authors.lastname: Kim
authors.forename: Jung-Hye
authors.initials: JH
authors.lastname: Shin
authors.forename: Jong Sang
authors.initials: JS
authors.lastname: Kim
authors.forename: Jeong Hwan
authors.initials: JH
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D004795:Enzyme Stability; D004926:Escherichia coli; D005340:Fibrinogen / Q000032:analysis / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D005340:Fibrinogen