Fig 5. Cleavage reactions by fluorescent recombinant protein substrates.

(A) Schematic representation of a recombinant fusion protein substrate and scheme of in-solution cleavage reactions. The TEV PR and Ty1 PR cleavage sites are colored by red and green, respectively. Cleavage site sequences are also shown for both proteases, asterisks indicate cleavage position. Red arrow shows cleavage by Ty1 PR, upon cleavage of the substrate (“S”), N- and C-terminal cleavage products (“N” and “C”, respectively) are produced. After enzymatic digestion, the cleavage products and uncleaved substrates can be separated by denaturing SDS-PAGE. Proteins can be visualized after in-gel renaturation of fluorescent proteins by blue light transillumination or by Coomassie staining. (B) Representative gel images are shown for substrates containing PR/IN, IN/RT, and Gag/PR cleavage sites, after cleavage reactions the bands were visualized in the polyacrylamide gels by blue light transillumination (upper gel images) and by Coomassie staining (lower gel images), as well. (C) The workflow of cleavage site identification, which includes a cleavage reaction with Ty1 PR, the separation of the cleavage fragments, and the digestion of N-terminal cleavage fragment by TEV PR. Resulted short fragments can be subjected to MALDI TOF-MS analysis. The recombinant substrate is identical with that one shown in figure part A, but here we show its immobilization to magnetic affinity beads (Ni-NTA). (D) The molecular weights (Da) of proteolytic fragments were calculated by ProtParam tools of ExPASy (available at https://web.expasy.org/protparam), and were compared to [M+H]⁺ values (Da) determined by MALDI-TOF MS. ^# denotes detection with low intensity.