PMID:8453373

Kim, JS and Raines, RT (1993) Ribonuclease S-peptide as a carrier in fusion proteins.Protein Sci. 2:348-56

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 Xa protease. The target was beta-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 (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).

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