Cloning of a Novel vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr

Abstract

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40°C, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.

Keywords: Bacillus subtilis; aprE; fibrinolytic activity; vpr.

Fig. 1. Coomassie Blue-stained gel (A) and fibrin zymogram (B) of supernatant from B. subtilis SJ4.

M, Dokdo-marker (EBM-1032, Elpis-Biotech., Korea). B. subtilis SJ4 was grown in LB broth for 96 h at 37°C. 1, 12 h; 2, 24 h; 3, 36 h; 4, 48 h; 5. 60 h; 6, 72 h; 7, 84 h; 8, 96 h. 10 μg protein concentrated by TCA precipitation was used for SDS-PAGE, and 1 μg protein without TCA precipitation was loaded for fibrin zymography. 10% acrylamide gels were used with 5% stacking gels.

Fig. 2. Alignment of amino acid sequence of mature VprSJ4 with homologous enzymes, Vpr from B. subtilis GP279 (M76590), and Vpr from B. subtilis KCTC 3014 (AY973268).

Amino acids different from each other are marked with an asterisk above.

Fig. 3. Purification of recombinant VprSJ4 from insoluble fraction.

M, Dokdo-marker (EBM-1034, Elpisbio, Korea); 1, insoluble fraction; 2, eluent at 100 mM imidazole concentration; 3, eluent at 200 mM imidazole concentration.

Fig. 4. SDS-PAGE and western blot of VprSJ4 purified from insoluble fraction.

(A) SDS-PAGE of purified VprSJ4 using a HiTrap IMAC FF column. (B) Western blot analysis of VprSJ4. M1, Dokdo-marker (EBM-1034); M2, Dokdo-marker (prestained, EBM-1032); 1, insoluble fraction from E. coli BL21(DE3) [pETvprSJ4] grown for 20 h after IPTG induction; 2, flow through; 3, eluent from column flushed with wash buffer (20 mM sodium phosphate, pH 7.4, 20 mM imidazole, and 0.5 M NaCl); 4, eluent from column flushed with elution buffer with 100 mM imidazole concentration. After color development of the PVDF membrane with alkaline phosphatase substrate solution, the bands were purple. The color tone of the scan result was adjusted to blue to enhance the contrast.

Fig. 5. Properties of Recombinant VprSJ4.

VprSJ4 eluted at 200 mM imidazole concentration was centrifuged using an Ultra-15 Centrifugal Filter (Amicon, 3 kDa, MWCO) to remove imidazole. Desalted VprSJ4 was used for studies on its properties. pH 7.0 was the optimum pH for the activity and VprSJ4 maintained higher activity at pH 6-10 (Fig. 5A). The optimum pH was 9.0 for partially purified Vpr from B. subtilis KCTC3014 [22]. The difference in optimum pH might be due to the different amino acid sequences or additional 4 amino acids of VprSJ4 at the C-terminus. VprSJ4 showed relative activity of 87.2, 91.4, 81.7, and 77.7% at pH 6, 8, 9, and 10, respectively. VprSJ4 showed no activity at pH 4 and below. The activity declined rapidly at pH above 10. During 6 h incubation, the activity decreased continuously at all pHs, and rapid inactivation occurred at pH 5 and below (Fig. 5B). The optimum temperature was 40°C at pH 7 (Fig. 5C), which was the same temperature reported for partially purified Vpr from B. subtilis KCTC3014 [22]. When the temperature was increased to 55°C and above, no activity was detected after 30 min incubation. During 3 h incubation, the activity decreased continuously, and especially decreased rapidly at 50°C and above. The results showed that VprSJ4 has moderate stabilities against pH and temperature.

Fig. 6. Growth (A) and fibrinolytic activities (B) of B. subtilis WB600 strains.

-●-, B. subtilis SJ4; -○-, B. subtilis WB600 [pHY300PLK]; -▼-, B. subtilis WB600 [pHYavSJ4]; -△-, B. subtilis WB600 [pHYaprESJ4].

Fig. 7. Coomassie Blue-stained gel (A) and fibrin zymogram (B) of culture supernatant from B. subtilis WB600 strains.

M, Dokdo-marker (EBM-1034); lanes 1-3, B. subtilis SJ4 culture supernatant at 24 h (1), 48 h (2), and 96 h (3); lanes 4-6, B. subtilis WB600 [pHYavSJ4] culture supernatant at 24 h (4), 48 h (5), and 96 h (6); lanes 7-9, B. subtilis WB600 [pHYaprESJ4] culture supernatant at 24 h (7), 48 h (8), and 96 h (9).