Peptide tags for a dual affinity fusion system.

Fusion protein systems can facilitate the purification of proteins produced by recombinant DNA technology (1-3). In these systems, a tag is fused to a target protein. The affinity of the tag for a specific ligand is exploited for the purification of the fusion protein. Here, we demonstrate that during affinity purification many truncated polypeptides carrying the affinity tag are copurified with the full-length fusion protein. We also show that use of a dual affinity fusion system allows isolation of homogeneous fusion protein. Finally, we describe short peptides that can serve as effective tags in a dual affinity fusion system. We recently developed a new fusion protein system in which the S-peptide of ribonuclease A (RNase A) was employed as an affinity tag (4). S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of RNase A by subtilisin. S-peptide binds S-protein with high affinity to form RNase S, which has full enzymatic activity. This specific interaction allowed the facile purification and detection of a fusion protein in which a modified S-peptide (DI4N SIS) was attached at the Nterminus of fj-galactosidase. One-step affinity chromatography under nondenaturing conditions yielded fusion protein that was >95% pure. Nevertheless, an extremely sensitive gel assay-zymogram electrophoresis-revealed that many truncated polypeptides carrying the D14N S15 tag were copurified with the fulllength fusion protein. This result was not unexpected, as the heterologous production of proteins in Escherichia coli often generates such truncated proteins, which are likely to be the products of proteolytic degradation. In a dual affinity fusion system, a different tag is attached to each end of the target protein. Affinity purification using each of these tags eliminates contamination from truncated polypeptides that contain only one tag. This approach has been described with protein tags (5-7). Proteins are undesirable tags, however, because their large size is likely to perturb properties of a target protein. Here, we describe the use of pep tides as tags for a dual affinity fusion system.