Intein-mediated purification systems

Fig. 1. (Continued) the N-terminus to the C-terminus starting with 1 (conserved residues Cys, Ser, rarely Ala). N-extein amino acids are denoted as negative numbers starting with -1 (X) at the N-terminal splice junction. The amino acids in the C-extein are numbered from the N-terminus to the C-terminus, beginning with h-1 (conserved residues Cys, Ser or Thr). Arrows indicate the direction of sequential numbering of residues. Conserved residues of inteins that are applied in intein-mediated purification systems are shown in the lower part of Fig. IB.

Fig. 3. Intein-mediated protein ligation. The IMPACT system allows affinity purification of proteins fused to an intein-CBD tag and their further isolation with a C-terminal thioester moiety (A), or an N-terminal cysteine (B). (A), N-terminal intein splicing for thioester isolation. Target protein (protein 1) is expressed in E. coli with C-terminally located intein-CBD tag. After specific binding to the chitin resin, the thiol reagent provokes the cleavage of the peptide bond between the target protein and the intein. Whereas the intein-CBD tag remains bound to the chitin resin, the protein thioester is eluted from the column. (B), C-terminal cleavage to obtain N-terminally...

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