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

Yao, Zhuang; Meng, Yu; Le, Huong Giang; Lee, Se Jin; Jeon, Hye Sung; Yoo, Ji Yeon; Kim, Hyun-Jin; Kim, Jeong Hwan

doi:10.4014/jmb.2006.06014

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

Journal of microbiology and biotechnology, v.30 no.11, 2020년, pp.1720 - 1728

Yao, Zhuang (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) , Meng, Yu (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) , Le, Huong Giang (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) , Lee, Se Jin (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) , Jeon, Hye Sung (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) , Yoo, Ji Yeon (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) , Kim, Hyun-Jin (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) , Kim, Jeong Hwan (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University)

Abstract ▼ AI-Helper

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℃, 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.

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참고문헌 (27)

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

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

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