Abstract
Bovine pancreatic ribonuclease (RNase) A, which consists of 124 amino acids, was synthesized in a conventional manner by assembling relatively small 30 peptide fragments of established purity, followed by applying a new deprotecting procedure with trifluoromethanesulfonic acid-thioanisole. After formation of disulfide bond by a glutathionemediated air-oxidation and subsequent two step purifications by affinity chromatography and ion-exchange chromatography, a protein with the full enzymatic activity of RNase A was obtained. The synthetic protein was subsequently crystallized from aqueous ethanol according to Kunitz. A totally synthetic enzyme with full RNase activity was thus obtained in a crystalline form for the first time.
