Cloning, Expression And Property Research Of Staphylococcus Aureus SLMWDSP And SPP2C

Dual-specificity protein phosphatases (DSPs) constitute a new family of protein tyrosine phosphatases (PTPs) characterized by the ability to dephosphorylate both phospho-tyrosyl residues and phospho-seryl/threonyl residues. They play key roles in the regulation of mitosis, signal transduction, extracellular stimulation and the cell cycle.Two novel dual specificity protein phosphatases named sLMWDSP (low molecular weight dual specificity phosphatase, Staphylococcus aureus) and sPP2C (protein phosphatase 2C, Staphylococcus aureus) were cloned from Staphylococcus aureus gene library. sLMWDSP contained 462 bp. The protein contained 154 amino acids and a dual specifity phosphatase catalytic domain. The molecular weight was 18 kDa, and pI was 4.49. sPP2C contained 741 bp. The protein contained 247 amino acids and a protein phosphatases 2C catalytic domain. The molecular weight was 26.1 kDa, and pI was 4.95. sLMWDSP and sPP2C were expressed in E.coli. Rossetta and purified by affinity chromatography.sLMWDSP and sPP2C show phosphatase activity towards pNPP which is a common synthetic protein phosphatase substrate. sLMWDSP shows phosphatase activity with a maximum value at pH 6.7 and 35 ℃ and is independent of metal ions; sPP2C is optimum at about pH 11.0 and 37 ℃ and Mn²⁺ and its phosphatase activity must be dependent of and is enhanced by bivalent metal ions. sLMWDSP can not be inhibited by okadaic acid and EDTA but obviously inhibited by vanadate sodium. However, sPP2C can be inhibited by different inhibitors such as EDTA, vanadate sodium, sodium pyrophosphate and sodium phosphate. Enzyme property research reveals that both of sLMWDSP and sPP2C show phosphatase activity towards oligopeptides containing pSer/Thr and pTyr, indicating that sLMWDSP and sPP2C are two novel protein phosphatases with dual substrate specificity.