RECOMBINANT HUMAN PANCREATIC RIBONUCLEASE PRODUCED IN ESCHERICHIA-COLI - IMPORTANCE OF THE AMINO-TERMINAL SEQUENCE

Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glut athione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucle olytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3',5'-adenosine revealing the distinctive ribonucleolytic ac tivity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal s equence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism sp ectra. The present study has revealed the important contribution of th e amino-terminal sequence of hRNase I to the characteristics of the pr otein. (C) 1995 Academic Press, Inc.