Figure 1. The purity of the GST-PTPα fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Figure 2. PTPα phosphatase activity was measured in a DELFIA® assay using the following reaction conditions: 25 mM HEPES, pH 7.2, 50 mM NaCl, 2.5 mM EDTA, 5 mM DTT, 65 ng/μl BSA, Substrate: Phospho-Poly (Glu-Tyr) Biotinylated Peptide #1586 at 1.5 μM, and 0.25 ng/μl PTPα.
Purified recombinant human PTPα (Met174-Lys802) phosphatase, supplied as a GST fusion protein.
Enzyme is supplied in 50 mM Tris-HCl, pH7.5; 150 mM NaCl, 0.25 mM DTT, 0.1mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol, 7 mM glutathione.Store at -80° C.Keep on ice during use.Avoid repeated freeze-thaw cycles.
The theoretical molecular weight of the GST-PTPα fusion protein is 96 kDa. The purified phosphatase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. PTPα phosphatase activity was determined using a DELFIA® assay [Fig.2].
The GST-phosphatase fusion protein was produced by expressing recombinant human PTPα (Met174-Lys802) (GenBank Accession No. NM_002836) with an amino-terminal tag in E. coli. The protein was purified by one-step affinity chromatography using glutathione-agarose.
PTPα (PTPRA) is a transmembrane receptor tyrosine phosphatase implicated in the regulation of Src family kinases during the G2 to mitosis entry point. Two identified splice variants differ in the size of the extracellular region; the shorter form appears to be ubiquitously expressed while the larger protein is more limited in distribution (1). The cytoplasmic region of PTPα contains two putative catalytic domains. One phosphatase domain (D1) exhibits catalytic activity while the other (D2) may regulate phosphatase activity by allowing receptor dimer formation (2,3). PTPα is a physiological regulator of Src and Src family kinases (4). Constitutive phosphorylation of the carboxy-terminal Tyr789 of PTPα is essential for dephosphorylation of Src at Tyr527. Phosphorylation of PTPα at this residue also allows binding of the Grb2 inhibitor, restricting PTPα activation of Src (5,6). PKC-mediated phosphorylation of the PTP at Ser180 and Ser204 also increases PTPα phosphatase activity (7).
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