Ribonuclease S-peptide as a carrier in fusion proteins

Protein Sci. 1993 Mar;2(3):348-56. doi: 10.1002/pro.5560020307.


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).

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • DNA, Recombinant / genetics
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Plasmids
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Ribonucleases / chemistry
  • Ribonucleases / genetics
  • Ribonucleases / metabolism*


  • Carrier Proteins
  • DNA, Recombinant
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • ribonuclease S-peptide
  • Ribonucleases