Figure 1. The purity of the GST-CD45 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Figure 2. CD45 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 CD45.
Purified recombinant human CD45 (Leu193-Ser1143) 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-CD45 fusion protein is 120 kDa. The purified phosphatase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. CD45 phosphatase activity was determined using a DELFIA® assay [Fig.2].
The GST-phosphatase fusion protein was produced by expressing recombinant human CD45 (Leu193-Ser1143)(GenBank Accession No. NM_080921) with an amino-terminal tail in E. coli. The protein was purified by one-step affinity chromatography using glutathione-agarose.
The protein phosphatase (PTP) receptor CD45 is a type I transmembrane protein comprised of a pair of intracellular tyrosine phosphatase domains and a variable extracellular domain generated by alternative splicing (1). The catalytic activity of CD45 is a function of the first phosphatase domain (D1) while the second phosphatase domain (D2) may interact with and stabilize the first domain, or recruit/bind substrates (2,3). CD45 interacts directly with antigen receptor complex proteins or activates Src family kinases involved in the regulation of T- and B-cell antigen receptor signaling (1). Specifically, CD45 dephosphorylates Src-family kinases Lck and Fyn at their conserved negative regulatory carboxy-terminal tyrosine residues and upregulates kinase activity. Conversely, studies indicate that CD45 can also inhibit Lck and Fyn by dephosphorylating their positive regulatory autophosphorylation site. CD45 appears to be both a positive and a negative regulator that conducts signals depending on specific stimuli and cell type (1). Human leukocytes including lymphocytes, eosinophils, monocytes, basophils and neutrophils express CD45, while erythrocytes and platelets are negative for CD45 expression (4).
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