Cloning of a Novel vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr.
作者： Zhuang Yao , Yu Meng , Huong Giang Le , Se Jin Lee , Hye Sung Jeon
关键词: Bacillus subtilis 、 Chemistry 、 Signal peptide 、 Recombinant DNA 、 Enzyme 、 Protease 、 Biochemistry 、 Escherichia coli 、 Affinity chromatography 、 Amino acid
摘要: 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 activity. In this cloned gene (vprSJ4) encoding protein, mature Vpr and minor protease secreted by species. vprSJ4 encodes preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) prosequence (132 aa). The (650 has predicted molecular weight 68,467.35. Unlike Vprs other B. strains, VprSJ4 4 additional (DEFA) at C-terminus. was overexpressed in Escherichia coli. PreproVprSJ4 localized inclusion bodies, subjected to vitro renaturation purification an affinity column. SDS-PAGE western blot showed autoprocessing preproVprSJ4 occurred smaller proteins were produced. optimum pH temperature recombinant 7.0 40°C, respectively. Kinetic parameters measured using artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression aprESJ4 pHY300PLK increased activity further 117% when compared single expression same vector WB600.
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