Figure 4. (A) The DnaK-PrP is efficiently cleaved by thrombin.
DnaK and DnaK-PrP were affinity purified using Ni-NTA chromatography. DnaK-PrP was incubated with thrombin for the indicated times. The proteins were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. (B) DnaK has enzymatic activity when fused to PrP. DnaK and DnaK-PrP were affinity purified by Ni-NTA chromatography. Equal amounts of each protein were used in autophosphorylation reactions for the indicated times. The products of the reactions were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue (top panel). The dried gel was exposed to a phosphorimager cassette to assess 32P incorporation (bottom panel).