Figure 1. The purity of the GST-SHP-1 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Figure 2. SHP-1 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 1 ng/μl SHP-1.
Purified recombinant full-length human SHP-1 phosphatase, supplied as a GST fusion protein.
Storage: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-1 fusion protein is 92 kDa. The purified phosphatase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. SHP-1 phosphatase activity was determined using a DELFIA® assay [Fig.2].
The GST-phosphatase fusion protein was produced by expressing recombinant human SHP-1 (Met1-Lys597) (GenBank Accession No. NM_080548) with an amino-terminal GST tag in E. coli. The protein was purified by one-step affinity chromatography using glutathione-agarose.
SHP-1 (PTPN6) is a non-receptor protein tyrosine phosphatase that is expressed primarily in hematopoietic cells. The enzyme is composed of two SH2 domains, a tyrosine phosphatase catalytic domain, and a carboxy-terminal regulatory domain (1). SHP-1 removes phosphates from target proteins to downregulate several tyrosine kinase-regulated pathways. In hematopoietic cells, the amino-terminal SH2 domain of SHP-1 binds to tyrosine phosphorylated erythropoietin receptors (EpoR) to negatively regulate hematopoietic growth (2). Overexpression of SHP-1 in epithelial cells results in dephosphorylation of the Ros receptor tyrosine kinase and subsequent downregulation of Ros-dependent cell proliferation and transformation (3). Following ligand binding in myeloid cells, SHP-1 associates with the IL-3R β chain and downregulates IL-3-induced tyrosine phosphorylation and cell proliferation (4). Because SHP-1 downregulates various proliferation pathways, SHP-1 is considered a potential tumor suppressor and angiogenesis regulator (5,6).
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