lambda Protein Phosphatase (λPPase)
Lambda protein phosphatase (λPPase) is a Mn2+ dependent protein phosphatase with activity to remove
phosphate groups from serine, threonine, tyrosine, and histidine residues. It is the 221 amino-acid product of
the ORF221 open reading frame of bacteriophage lambda (1). Recombinant λPPase is purified from a strain of
E. coli that carries the λPPase gene. λPPase has minimum 95% purity with a molecular weight of 25 kDa as
determined by SDS-PAGE and Coomassie Blue staining. It has no detectable protease and nuclease activity.
1 unit of λPPase hydrolyzes 1 nmol of p-nitrophenyl phosphate per minute at 30°C in assay buffer of 50 mM
HEPES pH 7.5, 100 mM NaCl. 2 mM DTT, and 1 mM MnCl2.
25 mM HEPES, pH 7.5, 100 mM NaCl, 0.1 mM MnCl2, 0.1 mM EGTA, pH 8.0, 2 mM DTT, 50% glycerol.
The Storage Buffer has no detergent.
Optimal dephosphorylation condition for a particular protein substrate must be determined empirically. The
phosphatase activity of λPPase varies on protein substrates. No special buffer is required to carry out the
dephosphorylation reaction. Buffers that keep protein substrates stable are recommended. Including 0.1-1
mM MnCl2 will help the dephosphorylation reaction. The dephosphorylation reaction can be carried out at
30oC or 4oC.
Initial dephosphorylation test can be done with 10,000 units of λPPase to 1 mg of target protein at the target
protein concentration of 1-2 mg/ml in a buffer suitable for the target protein at 4oC overnight or 30oC for 30
min. Often, regular SDS-PAGE does not separate phosphorylated and dephosphorylated target protein. Other
detection methods are required to access the dephosphorylation efficiency.
Dephosphorylation can be done at the same time of affinity tag removal during protein purification.
Inactivation of λPPase
λPPase can be heat inactivated at 65oC for 1 hour in the presence of 50 mM EDTA. It is also inhibited by
general protein phosphatase inhibitors such as vanadate.
λPPase has no affinity tag and cannot be easily removed.
Store at below -70oC. Shipped with dry ice.
1.Cohen, P.T.W. and Cohen, P. (1989) Biochem. J. 260:931–934.
2.Zhuo, S. et al. (1993) J. Biol. Chem. 268:17754–17761.