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Figure 2
Limited proteolysis and the search for potential protein domains in RepA. (A) SDS–PAGE (20% polyacrylamide) corresponding to 1/3 volume of the digestion of 30  g of purified RepA (-) with different proteases, either  -Chymotrypsin (Cy), Trypsin (T), Clostripain (Cl), Staphylococcal-V8 (V8) or Pronase (P), for 60 min. Protein molecular weight standards are shown (ST), together with their molecular masses (in kDa). (B) A list of the peptides that were identified after N-terminal sequencing of the same proteolysis reactions (2/3 volume) shown in (A). The N-terminal coordinate is indicated for each peptide [-2 corresponds to the GS sequence from the His6-tag, previous to the first RepA residue, that remains after thrombin-digestion; see (C)]. The expected molecular masses for the fragments are also shown [those corresponding to peptides visualized in (A) are in bold]. (C) Mapping the proteolytic cleavage sites (B) in the sequence of RepA protein. Dots are located every 10 amino acid residues. Lower-case numbers after the protease codes [as in (A)] rank the frequency of cleavage. Ovals encircle the two proteolytic hot-spots that define the existence of domains (dashed boxes) in RepA. Below the sequence of the protein, a secondary structure prediction (PHD) is shown. The box at the bottom further explains the symbols used in this figure.
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