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Highly Active and Removable AffinityTagged Lambda Phosphatase

ated target protein in the absence of interfering phosphatase.

This highly active lambda phosphatase was produced in Stratagene's Affinity protein expression and purification system. The lambda phosphatase gene was cloned into the pCALn vector as a fusion to an affinity tag, the calmodulinbinding peptide (CBP).5 The tag simplifies purification of the enzyme and allows the enzyme to be removed after dephosphorylation of the target protein. As the target protein may be a kinase, removal of the phosphatase is not only convenient but also essential for the subsequent determination of kinase activity.

Removal of Lambda Phosphatase Activity

The lambda phosphatase activity was specifically removed by treatment with Stratagene's Calmodulin (CaM) Affinity Resin. The efficiency of removing lambda phosphatase activity by binding to CaM resin was demonstrated by mixing Stratagene's autophosphorylated Epidermal Growth Factor Receptor Kinase Domain (EGFRKD) with Lambda Phosphatase and a 10% slurry of CaM resin in binding buffer (50 mM TrisHCl, pH 7.5; 150 mM NaCl; 10 mM beta-mercaptoethanol; 1 mM magnesium acetate; 1 mM imidazole, 2 mM CaCl2). Binding was carried out on ice for 10 minutes. The CaM resin was collected by lowspeed centrifugation (1600 rpm, 2 minutes, 4C). This binding procedure was repeated twice, and the supernatant was assayed for phosphatase activity by reaction with the chromogenic substrate, p-nitrophenyl phosphate (pNPP). After three consecutive resin treatments, the supernatant contained no detectable phosphatase activity as measured by dephosphorylation of the EGFRKD.

Assay of Lambda Phosphatase Activity

figure 1

To demonstrate the activity of Lambda Phosphatase, we used autophosphorylated EGFRKD as a protein substrate. Affinitytagged Lamb
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