Figure 1. The purity of the GST-PTPN7 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Figure 2. PTPN7 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 0.125 ng/μl PTPN7.
Purified recombinant full-length human PTPN7 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-PTPN7 fusion protein is 66 kDa. The purified phosphatase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. PTPN7 phosphatase activity was determined using a DELFIA(R) assay [Fig.2].
The GST-phosphatase fusion protein was produced by expressing recombinant human PTPN7 (Met1-Pro360) (GenBank Accession No. NM_002832) with an amino-terminal GST tag in E. coli. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Hematopoietic protein tyrosine phosphatase (HePTP, PTPN7) is a non-receptor protein tyrosine phosphatase that is expressed primarily in hematopoietic cells (1). The protein is made up of a short, amino-terminal extension that contains a conserved kinase interaction motif (KIM) and a carboxy-terminal protein tyrosine phosphatase domain (1,2). The presence of the kinase interaction motif allows PTPN7 to interact with MAPK family kinases, such as Erk1/2 and p38 MAP kinases (2,3). Reciprocal modification occurs between PTPN7 and Erk2, as the kinase phosphorylates PTPN7 at Thr45 and Ser72 while the phosphatase dephosphorylates Erk2 at Tyr185 (2). Down regulation of MAPK signaling follows removal of specific phospho-tyrosine resides from these MAPK kinases (3,4). Modification of MAPK family kinases by PTPN7 can regulate both kinase activity and localization (4).
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