The Yersinia protein tyrosine phosphatase (PTPase) Yop51, a C235R point mutation (Yop51*), and a protein lacking the first 162 amino acids at the NH2 terminus (Yop51*Δ162) have been overexpressed in Escherichia coli and purified to homogeneity through the use of CM Sephadex C25 cation exchange chromatography followed by Sephadex G-100 gel filtration. Greater than 50 mg of homogeneous Yop51* and Yop51*Δ162 can be obtained from a single liter of bacterial culture, whereas the same procedure yields only 5 mg of pure Yop51. Large, diffraction-quality crystals have been obtained for Yop51*Δ162. Size exclusion chromatography, sedimentation equilibrium, and enzyme concentration dependence experiments have established that the Yersinia PTPases exist and function as monomers in solution. Yop51 and Yop51* display identical UV, CD, and fluorescence spectra and have identical kinetic and structural stability properties. These full-length Yersinia PTPases have 31% α-helix, an emission maximum of 342 nm, a turn-over number of 1200 s-1 at pH 5.0, 30 °C, and an unfolding ΔG value of 6 kcal/mol at 25 °C. Yop51*Δ162 has very similar kinetic and fluorescence characteristics to the full-length molecules, whereas its CD and UV spectra show noticeable differences due to the elimination of 162 NH2-terminal residues. The Yersinia PTPases are by far the most active PTPases known, and their kinetic parameters are extremely sensitive to the ionic strength of reaction medium.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1992|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology