Expression, purification and crystallization of human phosphotyrosine phosphatase 1B.
Protein phosphotyrosine phosphatases are believed to be involved in the regulation of the activity of cellular proteins, such as receptor tyrosine kinases, by controlling their phosphorylation status. One of the best described and characterized protein of this class of enzymes is the phosphotyrosine phosphatase 1B. To obtain sufficient quantities for structural investigations, truncated forms of PTP1B encompassing the catalytic domain were over-expressed in Escherichia coli and purified to apparent homogeneity by conventional chromatography. The activity of these purified enzymes has been compared with the wild-type enzyme expressed in mammalian cells. By measuring the activities against p-nitrophenyl phosphate, the pH dependence of this activity, and responses to different modulators, it could be demonstrated that the truncated forms of PTP1B retained the same characteristics as the full-length mammalian enzyme, but are not subject to inhibition of enzymic activity mediated by the C-terminus. Due to their improved solubility, it can be assumed that the catalytic domains are advantageous for crystallization studies in comparison to the natural enzyme. In a screening for crystallization conditions, we obtained protein crystals indicating that the quality of the purified protein is sufficient for crystallographic studies.
Hoppe
E
E
Abteilung für Molekularbiologie, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Berne
P F
PF
Stock
D
D
Rasmussen
J S
JS
Møller
N P
NP
Ullrich
A
A
Huber
R
R
D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D011994:Recombinant Proteins; C008644:nitrophenylphosphate; D017027:Protein Tyrosine Phosphatases