| Ribonuclease S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of bovine pancreatic ribonuclease A (RNase A) by subtilisin. S-Peptide binds S-protein with high affinity to form RNase S, which has full enzymatic activity. We developed a novel protein fusion system for the purification and detection of proteins produced in Escherichia coli by employing S-peptide as an affinity tag. Any protein fused to S-peptide through recombinant DNA techniques can be purified by one-step affinity chromatography using an immobilized S-protein resin, and assayed by measuring ribonuclease activity following activation with S-protein.;Bovine seminal ribonuclease (BS-RNase) is a close homolog of RNase A, sharing 81% amino acid sequence identity. Unlike RNase A, BS-RNase is a dimer crosslinked by two disulfide bonds between Cys31 of one subunit and Cys32 of the other. At equilibrium, this dimer is a mixture of two distinct quaternary forms, M=M and MxM. In the major form, MxM, the N-terminal tail (equivalent to S-peptide) of one subunit stretches out from the body (equivalent to S-protein) of the same subunit, and interacts with the body of the other subunit. In the other form, M=M, such exchange does not occur. In addition to this intriguing quaternary structure, BS-RNase has extraordinary biological properties including aspermatogenic, antitumor, and immunosuppressive activities. To illuminate structure-function relationship of BS-RNase and RNase A, we synthesized and expressed a gene for BS-RNase in E. coli. Next, we prepared and characterized a series of BS-RNase mutants and molecular hybrids between BS-RNase and RNase A. All the hybrids we tested showed strong biological activities, which suggests that a receptor specific for BS-RNase may not exist. In contrast, C31S and C32S BS-RNases had a reduced fraction of MxM at equilibrium and decreased biological activities. These results indicate that the MxM form, which can remain as a dimer in the reducing environment of the cytosol, is responsible for the special biological properties of BS-RNase. |
