Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase.
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.
Zhang
Z Y
ZY
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Clemens
J C
JC
Schubert
H L
HL
Stuckey
J A
JA
Fischer
M W
MW
Hume
D M
DM
Saper
M A
MA
Dixon
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014508:Urea; D015007:Yersinia / Q000201:enzymology* / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014508:Urea; D017027:Protein Tyrosine Phosphatases