Figure 1. The purity of the GST-SHP-2 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Figure 2. SHP-2 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 0.06 ng/μl SHP-2.
Purified recombinant human SHP-2 (Gly246-Arg593) 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-SHP-2 fusion protein is 69 kDa. The purified phosphatase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. SHP-2 phosphatase activity was determined using a DELFIA(R) assay [Fig.2].
The GST-phosphatase fusion protein was produced by expressing recombinant human SHP-2 (Gly246-Arg593) (GenBank Accession No. NM_002834) with an amino-terminal GST tag in E. coli. The protein was purified by one-step affinity chromatography using glutathione-agarose.
SHP-2 (PTPN11) is a ubiquitously expressed, nonreceptor protein tyrosine phosphatase (PTP). It participates in signaling events downstream of receptors for growth factors, cytokines, hormones, antigens, and extracellular matrices in the control of cell growth, differentiation, migration, and death (1). Activation of SHP-2 and its association with Gab1 is critical for sustained Erk activation downstream of several growth factor receptors and cytokines (2). In addition to its role in Gab1-mediated Erk activation, SHP-2 attenuates EGF-dependent PI3 kinase activation by dephosphorylating Gab1 at p85 binding sites (3). SHP-2 becomes phosphorylated at Tyr542 and Tyr580 in its carboxy-terminus in response to growth factor receptor activation (4). These phosphorylation events are thought to relieve basal inhibition and stimulate SHP-2 tyrosine phosphatase activity (5). Mutations in the corresponding gene result in a pair of clinically similar disorders (Noonan syndrome and LEOPARD syndrome) that may result from abnormal MAPK regulation (6).
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