Posttranslational incorporation of noncanonical amino acids in the RNase S system by semisynthetic protein assembly.

Abstract

The unique ribonuclease S (RNase S) system, derived from proteolytic cleavage of bovine ribonuclease A (RNase A), consists of a tight complex formed by a peptide (amino acids 1-20) and a protein (21-124) part. These fragments, designated as S-peptide and S-protein, can be separated by two purification steps. By addition of synthetic S-peptide derivatives to the S-protein, semisynthetic RNase S is reassembled with high efficiency. Based on this peptide-protein complementation noncanonical amino acids can be easily introduced into a protein host. Here we describe the preparation of the S-protein from RNase A as well as the characterization of the reassembled semisynthetic RNase S complex. Complex formation can be monitored by RNase activity, circular dichroism, or fluorescence polarization. Structure-based enzyme design of the RNase S scaffold is possible based on high-resolution crystal structures of RNase S and its semisynthetic variants.