Protein Engineering to Exploit and Explore Bovine Secretory Ribonucleases

PROTEIN ENGINEERING TO EXPLOIT AND EXPLORE -----BOV~~EeR~~~~ELEAS~~~S====~==~~~---Jin-Soo Kim Under the supervision of Dr. Ronald T. Raines at the University of Wisconsin-Madison Ribonuclease 5-peptide (residues 1-20) and 5-protein (residues 21124) are the enzymatically inactive products of the limited digestion of bovine pancreatic ribonuclease A (RNase A) by subtilisin. 5-Peptide binds 5-protein with high affinity to form RNase 5, 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 Speptide as an affinity tag. Any protein fused to 5-peptide through recombinant DNA techniques can be purified by one-step affinity chromatography using an immobilized 5-protein resin, and assayed by measuring ribonuclease activity following activation with 5-protein. Bovine seminal ribonuclease (BS-RNase) is a close homolog of RNase A, sharing 81% amino acid sequence identity. Unlike RNase A, BSRNase 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 5-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 iv · -structure, BS--RNase-11.-as--**t-mGl'a-i-na-r-y-t>-i-o.lo.gicaL~ti:es:-::inrlndin-ag~~-------=---~-= 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.