Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases

We have developed a specific and efficient method for complete removal of p olyhistidine purification tags (HisTags) from the N-termini of target prote ins. The method is based on the use of the aminopeptidase dipeptidyl peptid ase I (DPPI), either alone or in combination with glutamine cyclotransferas e (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the HisTag i s cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain. Some sequences, howev er, are resistant to DPPI cleavage and a number of mature proteins have non substrate N-termini which protects them against digestion. For such protein s, HisTags composed of an even number of residues can be cleaved off by tre atment with DPPI alone. When the target protein is unprotected against DPPI , a blocking group is generated enzymatically from a glutamine residue inse rted between the HisTag and the target protein. A protein with a HisTag-Gln extension is incubated with both DPPI and GCT. As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented. The desired sequ ence is finally obtained by excision of the pyroglutamyl residue with PGAP. All the enzymes employed can bind to immobilized metal affinity chromatogr aphy (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our a minopeptidase-based method for specific excision of HisTags and use of subt ractive IMAC for removing processing enzymes. Typical recoveries were 75-90 % for the enzymatic processing and subtractive IMAC. The integrated process holds promises for use in large-scale production of pharmaceutical protein s because of a simple overall design, use of robust and inexpensive matrice s, and use of enzymes of either recombinant or plant origin. (C) 1999 Acade mic Press.