Cloning and expression of a gene encoding NTPase of Toxoplasma gondii (practical use for diagnosis)

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In this project, we defined that there were two NTPase isozymes (NTPase-I and NTPase-II) in the RH strain of Toxoplasma gondii and two genes encoding NTPase-I and NTPase-II of the RH strain and a gene encoding NTPase-II of the Beverly strain were cloned and the nucleotide sequences of these genes were determined. It was found that all the strains as tested have NTPase-II universally and only virulent strains have both isozymes by the enzyme kinetic studies and PCR gene amplification. These results will be published in May issue of Journal of Biological Chemistry, 1995. Above results suggested that a target isozyme to express in E.coli cells was NTPase-II which was present universally in all strains. Thus we constructed an expression plasmid including a gene encoding NTPase-II and tested gene expression in E.coli cell. Isopropyl beta-D-thiogalactopyranoside (IPTG) -inducible pGEMEX-1 and heat-inducible pPL-lambda were used as the expression vectors. It was confirmed that the E.coli cells treated with IPTG produced NTPase-II protein by SDS-polyacrylamide gel electrophoresis and following western blot analysis using anti-NTPase antibody and peroxidase conjugated antibody. Heat induction was a similar result. Although we succeeded to express NTPase-II protein in E.coli cells using IPTG and heat inducible plasmids, the efficiencies of both expression systems were insufficient because of a possibility that NTPase protein was toxic to E.coli cell. It is possible to use these E.coli cell lysates for a diagnostic ELISA method using a monoclonal antibody, however, more efficient expression system will be necessary to simplify the ELISA method. In this project, it was also found that NTPase gene was coding extra 25 aminoacids with some properties of a signal peptide. The data suggested that NTPase was a secretory enzyme. We found that NTPase was secreted form parasite to host cell actually. These results were published in Experimental Parasitology (79,301-311,1994) .