Based on mutagenesis studies using paramyosin as the target protein, Ser, Pro or Asp at the -1 position (the amino acid preceding the intein) blocks the cleavage When the C-terminus of a target protein contains an unfavorable residue, additional residue(s) such as Tyr (the native residue preceding the Mxe GyrA intein), may be inserted between the target protein and the N-terminus of the intein to improve controlled cleavage. However, the cleavage efficiency may vary when a different target protein is fused to the intein.