Ribonuclease S‐peptide as a carrier in fusion proteins

Abstract

S‐peptide (residues 1–20) and S‐protein (residues 21–124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S‐peptide binds S‐protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S‐peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four‐residue sequence recognized by factor X_a protease. The target was β‐galactosidase. The interaction between the S‐peptide portion of the fusion protein and immobilized S‐protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S‐protein. S‐peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (≥15 residues), a tunable affinity for ligand (K_d ≥ 10⁻⁹ M), and a high sensitivity of detection (≥10⁻¹⁶ mol in a gel).