Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase

Zhong-Yin Zhang, James C. Clemens, Heidi L. Schubert, Jeanne A. Stuckey, Mark W F Fischer, Daniel M. Hume, Mark A. Saper, Jack E. Dixon

Research output: Contribution to journalArticle

166 Citations (Scopus)

Abstract

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 languageEnglish (US)
Pages (from-to)23759-23766
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number33
StatePublished - Nov 25 1992
Externally publishedYes

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Yersinia
Recombinant proteins
Protein Tyrosine Phosphatases
Recombinant Proteins
Purification
Fluorescence
Kinetics
Size exclusion chromatography
Chromatography
Ionic strength
Sedimentation
Kinetic parameters
Escherichia coli
Gel Chromatography
Cations
Ion exchange
Diffraction
Monomers
Gels
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Zhang, Z-Y., Clemens, J. C., Schubert, H. L., Stuckey, J. A., Fischer, M. W. F., Hume, D. M., ... Dixon, J. E. (1992). Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase. Journal of Biological Chemistry, 267(33), 23759-23766.

Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase. / Zhang, Zhong-Yin; Clemens, James C.; Schubert, Heidi L.; Stuckey, Jeanne A.; Fischer, Mark W F; Hume, Daniel M.; Saper, Mark A.; Dixon, Jack E.

In: Journal of Biological Chemistry, Vol. 267, No. 33, 25.11.1992, p. 23759-23766.

Research output: Contribution to journalArticle

Zhang, Z-Y, Clemens, JC, Schubert, HL, Stuckey, JA, Fischer, MWF, Hume, DM, Saper, MA & Dixon, JE 1992, 'Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase', Journal of Biological Chemistry, vol. 267, no. 33, pp. 23759-23766.
Zhang, Zhong-Yin ; Clemens, James C. ; Schubert, Heidi L. ; Stuckey, Jeanne A. ; Fischer, Mark W F ; Hume, Daniel M. ; Saper, Mark A. ; Dixon, Jack E. / Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 33. pp. 23759-23766.
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