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Updated: May 3, 2026
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Published on: May 30, 2017
Domain isolation, expression, purification and proteolytic activity of the metalloprotease PrtV from Vibrio cholerae
Aaron Edwin1, Christin Grundström1, Sun N Wai2
1Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden; Umeå Centre for Microbial Research (UCMR), Umeå University, SE-901 87 Umeå, Sweden.
Abstract:
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 (102 kDa) 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 25 kDa fragment (residues 581-839). The soluble fusion proteins were captured with Ni²⁺ affinity chromatography, and subsequently cleaved with tobacco etch virus protease. Purification protocols yielded ∼10-15 mg of pure protein from 1L of culture. Proper folding of the shorter domains was confirmed by heteronuclear NMR spectra recorded on ¹⁵N-labeled samples. A modified protocol for the native purification of the secreted 81 kDa pro-protein of PrtV is provided. Proteolytic activity measurements suggest that the 37 kDa catalytic metalloprotease domain alone is sufficient for activity.
Insights
Researchers developed methods to express and purify key fragments of Vibrio cholerae metalloprotease PrtV. This facilitates structural studies of the bacterial invasion protein.
Area of Science:
- Microbiology
- Structural Biology
- Protein Chemistry
Background:
- The metalloprotease PrtV from Vibrio cholerae is crucial for host cell invasion.
- PrtV is a large, multidomain protein belonging to the M6 protease family, requiring high purity and solubility for structural studies.
- Previous recombinant expression attempts of PrtV fragments yielded low solubility and expression.
Purpose of the Study:
- To develop methods for recombinant expression and purification of soluble PrtV fragments.
- To identify specific domains of PrtV suitable for structural and functional analysis.
- To establish protocols for obtaining pure PrtV protein for further research.
Main Methods:
- Parallel cloning and expression of various fusion-tagged PrtV fragments in Escherichia coli.
- Ni²⁺ affinity chromatography for capturing soluble fusion proteins.
- Tobacco etch virus protease cleavage for tag removal and protein purification.
- Heteronuclear NMR spectroscopy for confirming proper folding of purified domains.
- Development of a modified protocol for native purification of the secreted pro-protein.
Main Results:
- Over 100 constructs were tested, with three yielding well-expressing protein products: the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25 kDa fragment (residues 581-839).
- Purification yielded approximately 10-15 mg of pure protein per liter of culture.
- NMR confirmed the proper folding of the expressed domains.
- A modified protocol for purifying the 81 kDa pro-protein was established.
- The 37 kDa catalytic metalloprotease domain was found to be sufficient for activity.
Conclusions:
- Successful recombinant expression and purification of key PrtV fragments were achieved, overcoming previous limitations.
- The identified soluble domains are suitable for structural and functional characterization.
- The developed protocols enable the production of sufficient pure PrtV protein for detailed studies.
- These findings pave the way for understanding the role of PrtV in Vibrio cholerae pathogenesis.
