Figure 4.

The proteolytically inactive variant His-ProtC₂₃₀₇A-Pol* does not release His-Prot. (A) The insoluble fraction of a lysate from induced cultures of E. coli transformed with the plasmid pet11a-His-ProtC₂₃₀₇A-Pol* was solubilized in 8 M urea buffer and subjected to IMAC. The individual imidazole elution fractions were resolved via SDS-PAGE and stained. Note the single protein species with an apparent molecular mass of 60 kDa that specifically eluted with increasing imidazole concentrations and the absence of a 35 kDa band (His-Prot). (B) Refolded IMAC-purified His-Prot-Pol* and His-ProtC₂₃₀₇A-Pol* were resolved side by side in SDS-PAGE and analyzed via Western blot using a His-tag-specific antibody. His-Prot of 35 kDa is not formed in the case of the inactivated variant His-ProtC₂₃₀₇A-Pol*. However, a weak band at 25 kDa (marked with an arrowhead in (A) and (B)) appeared, probably representing a product of degradation after prolonged expression time.