Figure 1. The purity of the GST-PTPN13 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Figure 2. PTPN13 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 PTPN13.
Purified recombinant human PTPN13 (Asp2169-Lys2485) Phosphatase, supplied as a GST fusion protein.
Enzyme is supplied in 20 mM MOPS, pH7.5; 50 mM NaCl, 0.25 mM DTT, 0.1 mM PMSF, 30% 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-PTPN13 fusion protein is 61 kDa. The purified phosphatase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. PTPN13 Phosphatase activity was determined using a DELFIA(R) assay [Fig.2].
The GST-phosphatase fusion protein was produced by expressing recombinant human PTPN13 (Asp2169-Lys2485) (GenBank Accession No. NM_080683) with an amino-terminal GST tag in E. coli. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Non-receptor protein tyrosine phosphatase 13 (PTPN13) is also known as Fas-associated phosphatase-1 (FAP-1) because it associates with the regulatory domain of the Fas receptor and negatively regulates Fas signaling (1). PTPN13 protein contains: an N-terminal kinase non-catalytic C-lobe domain (KIND); followed by a Four-point-one/Ezrin/Radixin/Moesin (FERM) domain; five PDZ domains; and a C-terminal phosphatase domain (2,3). The function of the KIND domain is not clear, while the FERM domain is thought to bind cell surface receptors and help target PTPN13 to the plasma membrane. The PDZ domains may help PTPN13 bind selectively to target proteins and maintain substrate specificity (2). Dephosphorylation of Fas receptor by PTPN13 reduces the presence of the receptor at the cell surface and inhibits Fas-mediated apoptosis (4). Both Fas receptor and its inhibitor are regulated through NF-κB signaling. Activated Fas receptor induces NF-κB signaling, which promotes expression of the Fas inhibitor PTPN13 (5). Induced overexpression of PTPN13 in vitro blocks IRS-1/PI3K/Akt signaling by dephosphorylating IRS-1, leading to reduced cell survival and induction of apoptosis (6). Mutations in the corresponding PTPN13 gene and abnormal phosphatase activity are associated with colorectal cancer, suggesting that PTPN13 plays an important role in regulating cancer cell proliferation (7). Similar studies indicate that PTPN13 may act as a tumor suppressor in hepatocellular carcinoma as inhibition of PTPN13 phosphatase activity results in increased cancer cell proliferation (8).
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