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Title
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Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase.
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Authors
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Z.Y.Zhang,
J.C.Clemens,
H.L.Schubert,
J.A.Stuckey,
M.W.Fischer,
D.M.Hume,
M.A.Saper,
J.E.Dixon.
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Ref.
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J Biol Chem, 1992,
267,
23759-23766.
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PubMed id
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Abstract
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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*delta 162) 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*delta 162 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*delta 162. 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% alpha-helix, an emission
maximum of 342 nm, a turn-over number of 1200 s-1 at pH 5.0, 30 degrees C, and
an unfolding delta G value of 6 kcal/mol at 25 degrees C. Yop51*delta 162 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.
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