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Domain isolation, expression, purification and proteolytic activity of the metalloprotease PrtV from Vibrio cholerae
Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
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2014 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 96, 39-47 p.Article in journal (Refereed) Published
Abstract [en]

The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102kDa) multidomain pre-pro-protein that so far has only been expressed in V. cholerae. Structural studies require high amounts of soluble protein with high purity. Previous attempts for recombinant expression have been hampered by low expression and solubility of protein fragments. Here, we describe results from parallel cloning experiments in Escherichia coli where fusion tagged constructs of PrtV fragments were designed, and protein products tested for expression and solubility. Of more than 100 designed constructs, three produced protein products that expressed well. These include the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25kDa fragment (residues 581-839). The soluble fusion proteins were captured with Ni(2+) affinity chromatography, and subsequently cleaved with tobacco etch virus protease. Purification protocols yielded ∼10-15mg of pure protein from 1L of culture. Proper folding of the shorter domains was confirmed by heteronuclear NMR spectra recorded on (15)N-labeled samples. A modified protocol for the native purification of the secreted 81kDa pro-protein of PrtV is provided. Proteolytic activity measurements suggest that the 37kDa catalytic metalloprotease domain alone is sufficient for activity.

Place, publisher, year, edition, pages
Elsevier, 2014. Vol. 96, 39-47 p.
Keyword [en]
Vibrio cholerae, Metalloprotease, PrtV, Fusion tag, Expression screen, Escherichia coli
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-87821DOI: 10.1016/j.pep.2014.01.012ISI: 000332906300007PubMedID: 24492010OAI: oai:DiVA.org:umu-87821DiVA: diva2:711645
Funder
Swedish Research Council, K2013-67X-13001-15-3
Available from: 2014-04-10 Created: 2014-04-10 Last updated: 2017-05-10Bibliographically approved
In thesis
1. Structural and functional studies of the secreted metalloprotease PrtV from Vibrio cholerae
Open this publication in new window or tab >>Structural and functional studies of the secreted metalloprotease PrtV from Vibrio cholerae
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cholera, an acute diarrheal diseases caused by the intestinal infection of the pathogenic bacterium Vibrio cholerae, continues to be a global killer in the world today. PrtV, a secreted zinc metalloprotease, is a potent cytotoxic virulence factor of V. cholerae. The 102 kDa full length multi-domain PrtV protein undergoes several N and C terminal modifications before being secreted as a 81 kDa pro-protein. The activation of the pro-protein is calcium dependent. The removal of calcium triggers auto-proteolysis to give a stable active protease with the catalytic zinc binding domain. The aim of the thesis was to study the structure and function of the PrtV protein. The results from paper I, identified the end product of the maturation of PrtV as the stable 37 kDa M6 active domain, and not a 55 kDa complex as reported earlier. Results also showed the this 37 kDa active M6 domain alone was sufficient for catalytic activity. A revised model for the maturation of PrtV was proposed. Individual domains were isolated from the PrtV protein by domain phasing methods. This included the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25 kDa fragment (residues 589-839). The isolated domains were recombinantly over expressed as fusion proteins to increase expression and solubility. The PKD1 domain was purified to homogeneity and crystallized. The structure of the PKD1 domain reported in paper II, was solved by X-ray crystallography at an atomic resolution of 1.1 Å. From the structure, a previously unknown calcium binding site was identified at the N-terminal of the PKD1 domain. The structure also revealed two conformations for the PKD1 domain depending on free or bound calcium. From the structure, a function of the PKD1 domain as a protector of the cleavage site in the linker region between the M6 domain and the PKD1 domain in the presence of calcium was elucidated. A new model for the activation of PrtV was given. In paper III, the structure of the N-terminal domain solved by NMR spectroscopy was reported. The structure revealed two well defined helices but a third predicted helix was found to be unstructured.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2014. 44 p.
Keyword
Cholera, Vibrio cholerae, virulence factors, PrtV, X-ray crystallography, NMR
National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-84553 (URN)978-91-7459-793-6 (ISBN)
Public defence
2014-01-31, KBC-huset, KB3A9, Umeå universitet, Umeå, 14:00 (English)
Opponent
Supervisors
Available from: 2014-01-10 Created: 2014-01-08 Last updated: 2014-05-16Bibliographically approved

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Edwin, AaronGrundström, ChristinWai, Sun NyuntÖhman, AndersStier, GunterSauer-Eriksson, A Elisabeth
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