Gene Clone, Expression And Characterization Of A Novel Extracellular Protein Phosphatase From Metarhizium Anisopliae Strain CQMa102

Metarhizium anisopliae, one of the entomopathogenic fungi which are largestly studied and applied, extracts many enzymes related with infecting the host.It was reported that a novel extracellular protein tyrosine phosphatases (PTPase) secreted by entomogenic fungus Metarhizium anisopliae could dephosphorylate the signal transduction substance (Toll-like receptor and trans-Golgi p230) of locust humoral immunity specifically in vitro, which implied that the fungus might interfere with the immune defense of locust. We found a novel sequence when studied PTPase that constituted by different Pre-mRNA splicing of hnRNA of PTPase. The putative amino acid sequence of the phosphatase shares identity with PTPase (GenBank No. DQ302474, No. EFY93239). It contains HCX5RS peptide, and according to the characterization, we imagine maybe the production of the sequence is a novel PTPase. However, we know nothing about its framework, biochemical characters and functions. Therefore, the objectives of this research were to clone the novel sequence from M. anisopliae CQMa102, to express the novel protein tyrosine phosphatase in a methylotrophic Pichia pastoris, and to characterize the PTPase, to provide material base for further study of the effect of PTPase on immune defense of locust.To generate enough active protein for studying its biochemical characters to provide reference for further studies of the functions of this phosphatase, we constructed recombinant plasmid were then transformed into Pichia pastoris KM71by electroporation to obtain the recombinant strain9k-PTPase. After induction in buffered methanol-complex medium (BMMY), SDS-PAGE and western blotting indicated that the recombinant9k-PTPase had expressed target protein with apparent molecular weight and pI to be around91kDa and6.88successfully. We selected the methanol concentration1%after120h incubation as the optimum inductive strategies after optimizing the fermentive condition. Express the protein cosmically under the optimum fermentive condition, the culture filtrate of9k-PTPase was concentrated, and then purified by HisTrap affinity column. The purified protein, showing high purity and homogeneity, was found to be phosphatase and its pI around6.88after analysis of IEF-PAGE. The main results of analysis of phosphatase character as follows:(1) The optimum temperature and pH for phosphatase activity against5mM O-phospho-L-tryosine were50℃and pH5.5.(2) The phosphatase activity against pTyr was strong significantly than other substance which indicates that the phosphatase is protein phosphatase.(3) Phosphatase activities of the purified enzyme against pNPP were not affected significantly by adding specific inhibitor of serine/threonine protein phosphatase and alkaline phosphatase, such as EDTA, which indicated that the phosphatase is neither serine/threonine protein phosphatase nor alkaline phosphatase; The experimental ion including Ca2+、Mg2+, which were known as activator of alkaline phosphatase, inhibited the activity; However, Phosphatase activities of the purified enzyme against pNPP were inhibited significantly by adding specific inhibitor of PTPase, such as orthovanadate, tungstate and Zn2+. But there is not direct evidence that the protein phosphatase is protein tyrosine phosphatase. Because of the results above and its activity against pThr, pSer and pNPP, we think the phosphatase is protein phosphatase with double specific activities.(4) Phosphatase activities of the purified enzyme against pNPP were inhanced by adding MnSO4and low concentration (1mM) of Fe2+, Ba2+and Ca2+, activity would be inhibited when the concentration increase; Phosphatase activities of target protein were inhibited significantly by Zn2+, Ag+, Cu2+while no obvious effect on phosphatase activity against pNPP was observed when Mg2+was used.