Expression and identification of a minor extracellular fibrinolytic enzyme (Vpr) from Bacillus subtilis KCTC 3014 |
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Authors: | Nack-Shick Choi Dong-Min Chung Chan-Sun Park Keug-Hyun Ahn Joong Su Kim Jae Jun Song Seung-Ho Kim Byung-Dae Yoon Min-Soo Kim |
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Affiliation: | 1.Institute Bioindustry Research Center,Korea Research Institute of Bioscience and Biotechnology (KRIBB),Jeungeup,Korea;2.Systemic Proteomics Research Center,Korea Research Institute of Bioscience and Biotechnology (KRIBB),Daejon,Korea;3.Enzyme Based Fusion Technology Research Team,Korea Research Institute of Bioscience and Biotechnology (KRIBB),Jeungeup,Korea |
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Abstract: | Previously, three extracellular proteases, Vpr, PepT, and subtilisin were identified from Bacillus subtilis KCTC 3014. To confirm the activity of Vpr, two recombinant Vpr proteins, full Vpr with TTG (pGST-fTTG-Vpr) and full Vpr with ATG (pGST-fATG-Vpr) as an initiation codon were expressed using a pGEX-2T vector encoding glutathione S-transferase (GST) in Escherichia coli. Vpr was produced in two forms, occurring as four spots on a 2-DE gel, 68 and 75 kDa proteins with similar pI values (4.0 ∼ 4.5). Activity was detected in a fibrin zymography at the expected molecular size of 68 kDa (mature form) processed from full Vpr. However, the recombinant 75 kDa of GST-fVpr did not exhibit activity. Replacement of the TTG codon with ATG led to 1.9-fold increased enzyme activity in 68 kDa. Interestingly, the expression of GSTVpr resulted in the proteolytic degradation of the protein and no GST fusion Vpr protein was detected. |
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