Figure 3.

Purification and general characterization of the recombinantly expressed G. intestinalis thymidine kinase.A, SDS-PAGE showing the purified fusion construct of His-tagged protein Z attached to the thymidine kinase (HisZ-TK). Lane 1 shows the full-length construct after nickel agarose chromatography (theoretically 52 kDa), lane 2 shows the same protein after cleavage, and lane 3 shows the purified nontagged thymidine kinase (TK, theoretically 42 kDa). The final nontagged protein was collected in the flow-through after cleavage and repurification on nickel agarose to remove noncleaved protein, HisZ (10 kDa, not visible in the gel), and His-tagged TEV protease (27 kDa). B, the effect of different salts on thymidine kinase enzyme activity. Standard enzyme assay conditions were used in the assays, and the thymidine concentration was 0.2 mM. The results in (B) represent the average of three independent experiments with standard errors indicated. C, mass photometry analysis of G. intestinalis thymidine kinase showed that it is a monomer. The measured molecular mass was close to the theoretical mass of 42 kDa. TEV, tobacco etch virus.