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Highly Active and Removable AffinityTagged Lambda Phosphatase

Dualspecificity Lambda Phosphatase useful for dephosphorylation serine, threonine or tyrosine residues

Danny Q. Hoang * Mary Ellen Simcox
Stratagene Cloning Systems, Inc.

Stratagene's recombinant Lambda Phosphatase is a dualspecificity protein phosphatase that has been produced as a fusion to Stratagene's novel affinity tag, the calmodulinbinding peptide. The tag simplifies purification of the enzyme and allows the enzyme to be removed subsequent to in vitro dephosphorylation of substrate proteins. The activity of the target protein can then be assayed in the absence of phosphatase activity. This feature is an essential advantage when the substrate protein itself is a protein phosphatase or protein kinase.

Protein phosphorylation governs many biological processes, including metabolism, gene expression, cytoskeletal architecture, cell adhesion and the cell cycle.1 The level of protein phosphorylation in cells is modulated by two large families of enzymes, the protein kinases and the protein phosphatases. Kinases and phosphatases are classified according to their substrate specificities as tyrosine, serine/ threonine or dualspecificity enzymes.2,3

Lambda phosphatase is a dualspecificity phosphatase4 that is useful for dephosphorylating proteins phosphorylated on serine, threonine or tyrosine residues. The enzyme can be used to determine whether a protein of interest is phosphorylated by analyzing the protein by SDSPAGE, plus and minus phosphatase treatment. Dephosphorylation results in a significant shift in electrophoretic mobility. In addition to this general application, Stratagene's novel Lambda Phosphatase is readily removable; binding to calmodulin affinity resin completely eliminates lambda phosphatase activity. By using Stratagene's Lambda Phosphatase, investigators are able to assay the activity of a dephosphoryl 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 da Phosphatase efficiently dephosphorylated the autophosphorylated tyrosines of EGFRKD (figure 1). Using 45 or 112 units, Lambda Phosphatase completely dephosphorylated 100 ng and 500 ng of EGFRKD (figure 1; lanes 5, 6, 8 and 9). Using a competitor's lambda phosphatase, we observed complete dephosphorylation only when 50 ng of EGFRKD was assayed (figure 1, lane 1). When more units of the competitor's lambda phosphatase were used under the same assay conditions, incomplete dephosphorylation of 100 ng and 500 ng of EGFRKD (lanes 2 and 3) is observed. As positive controls, 500 ng (lane 4) and 100 ng (lane 7) of autophosphorylated EGFRKD were loaded onto the gel and detected by immunoblotting with an antibody to phosphotyrosine, the PY20 monoclonal antibody.

figure 2

After reaction of EGFRKD with Lambda Phosphatase and treatment with the CaM affinity resin to remove the phosphatase, the tyrosine kinase (TK) activity of the EGFRKD was assayed. The ability to assay enzymatic activity of a protein substrate following dephosphorylation and phosphatase removal is a key feature of CBPtagged Lambda Phosphatase. Because many of the proteins that are phosphorylated in cells are kinases, the effect of phosphorylation on their own kinase activity is of great interest. The kinase activity of EGFRKD, as measured by autophosphorylation, was assayed after dephosphorylation by Lambda Phosphatase. A small loss of the EGFRKD due to nonspecific binding to the resin was observed (figure 2, lane 2). The amount of EGFRKD kinase activity is decreased in the presence of competing lambda pho sphatase (figure 2, lane 3). The tyrosine kinase activity of EGFRKD was not adversely affected by one or two CaM resin treatments (figure 2, lanes 4 and 5).


The data presented here demonstrate both the efficient dephosphorylation of substrate by Lambda Phosphatase and the removal of Lambda Phosphatase by CaM affinity resin treatment. Assay of target protein activity after dephosphorylation and phosphatase removal is also shown. These experiments illustrate the usefulness of Stratagene's affinitytagged Lambda Phosphatase for investigating the effects of phosphorylation on substrate protein binding and enzymatic activities.


  1. Pawson, T. (1994) FASEB J. 8: 11121113.

  2. Hunter, T. (1987) Cell 50: 823829.

  3. Guan, K.L., and Dixon, J.E. (1991) J. Biol. Chem. 266: 1702617030.

  4. Zhou, S., et al. (1993) J. Biol. Chem. 268: 1775417761.

  5. StofkoHahn, R.E., Carr, D.W., and Scott, J.D. (1992) FEBS Lett. 302: 274278.



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