Recombinant expression and purification of an enzymatically active cysteine proteinase of the protozoan parasite Entamoeba histolytica.

摘要: Cysteine proteinases and in particular cysteine proteinase 5 (EhCP5) of Entamoeba histolytica are considered important for ameba pathogenicity. To study EhCP5 more detail a protocol was elaborated to produce considerable amounts the enzyme its active form. The protein expressed Escherichia coli as histidine-tagged pro-enzyme purified homogeneity under denaturing conditions presence guanidine-HCl using nickel affinity chromatography. Renaturation performed by 100-fold dilution buffer containing reduced oxidized thiols, which led soluble but enzymatically inactive pro-enzyme. Further processing activation achieved 10 mM DTT 0.04% SDS at 37 degrees C. Recombinant (rEhCP5) indistinguishable from native E. lysates. Both runs SDS-PAGE reducing nonreducing positions corresponding 27 29 kDa, respectively, had same pH optima displayed similar specific activity against azocasein. Moreover, both enzymes were broad spectrum biological synthetic substrates such mucin, fibrinogen, collagen, human hemoglobin, bovine serum albumin, gelatin, IgG, Z-Arg-Arg-pNA, Z-Ala-Arg-Arg-pNA, not Z-Phe-Arg-pNA. identity rEhCP5 confirmed inhibition with inhibitors. In contrast, various compounds known specifically inhibit aspartic, metallo, or serine no effect on activity.