prescission

Moreover, other endopeptidases (e.g., PreScission and Sortase A) and exopeptidases (e.g., DAPase, Aeromonas aminopeptidase, aminopeptidase M, and carboxypeptidase A and B) were described exhaustively for the removal of affinity tags from recombinant proteins [1, 52].

PreScission protease is a GST tagged human rhinovirus (HRV) type 14 3C protease and specifically recognizes the amino acid sequence LEV[LFQ[down arrow]GP.bar] or its subset of sequences which include the core amino acid sequence (underlined), cleaving between the glutamine and glycine residues [72,73].

The created IA-2icpGEX plasmid contained a linker providing a cleavage site for PreScission Protease (Amersham Biosciences) between GST and IA2ic.

For purification of IA-2ic, GST-IA-2ic in 9 mL of cleared lysate (above) was bound to a Glutathione Sepharose column (bed volume, 3 mL), washed with cleavage buffer (50 mmol/L Tris-HCI, 150 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, pH 7.0), and cleaved in the column with 240 U of PreScission Protease for 16-18 h at 4[degrees]C.

GST-IA-2ic in cleared bacterial lysate was bound to a Glutathione Sepharose column, washed, and either eluted with reduced glutathione as the intact fusion protein or cleaved in the column with PreScission Protease, giving free IA-2ic in the eluate.