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1H, 15N and 13C Backbone Assignments and Secondary Structures of C-ter100 Domain of Vibrio Extracellular Metalloprotease Derived from Vibrio vulnificus

  • Yun, Ji-Hye ;
  • Kim, Hee-Youn ;
  • Park, Jung-Eun ;
  • Cheong, Hae-Kap ;
  • Cheong, Chae-Joon ;
  • Lee, Jung-Sup ;
  • Lee, Weon-Tae
  • Received : 2012.06.26
  • Accepted : 2012.07.06
  • Published : 2012.10.20

Abstract

Vibrio extracellular metalloprotease (vEP), secreted from Vibrio vulnificus, shows various proteolytic function such as prothrombin activation and fibrinolytic activities. Premature form of vEP has an N-terminal (nPP) and a C-terminal (C-ter100) region. The nPP and C-ter100 regions are autocleaved for the matured metalloprotease activity. It has been proposed that two regions play a key role in regulating enzymatic activity of vEP. Especially, C-ter100 has a regulatory function on proteolytic activity of vEP. C-ter100 domain has been cloned into the E. coli expression vectors, pET32a and pGEX 4T-1 with TEV protease cleavage site and purified using gel-filtration chromatography followed by affinity chromatography. To understand how C-ter100 modulates proteolytic activity of vEP, structural studies were performed by heteronuclar multi-dimensional NMR spectroscopy. Backbone $^1H$, $^{15}N$ and $^{13}C$ resonances were assigned by data from standard triple resonance and HCCH-TOCSY experiments. The secondary structures of vEP C-ter100 were determined by TALOS+ and CSI software based on hydrogen/deuterium exchange. NMR data show that C-ter100 of vEP forms a ${\beta}$-barrel structure consisting of eight ${\beta}$-strands.

Keywords

Vibrio extracellular metalloprotease (vEP);Secondary structure;NMR;Cloning;Purification

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Acknowledgement

Supported by : NRF