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KMID : 1094720100150030446
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Biotechnology and Bioprocess Engineering
2010 Volume.15 No. 3 p.446 ~ p.452
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Expression and Identification of a Minor Extracellular Fibrinolytic Enzyme (Vpr) from Bacillus subtilis KCTC 3014
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Choi Nack-Shick
Kim Min-Soo
Park Chan-Sun
Song Jae-Jun
Yoon Byung-Dae
Kim Joong-Su
Chung Dong-Min
Ahn Keug-Hyun
Kim Seung-Ho
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Abstract
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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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KEYWORD
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Bacillus subtilis, mass spectrometry, UUG start codon, Vpr, zymography
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