Production of engineered human pancreatic ribonucleases, solving expression and purification problems, and enhancing thermostability

Human pancreatic ribonuclease, the homolog of bovine pancreatic ribonucleas e, has a significant therapeutic potential. Its study has been hindered by the difficulty of obtaining the enzyme in a pure and homogeneous form, eith er from human source or using heterologous expression. Engineering of diffe rent variants of human pancreatic ribonuclease has allowed us to study and overcome some problems encountered during its heterologous production in an Escherichia coli system and its purification from inclusion bodies. The 5' -end region of the mRNA that encodes the enzyme is critical for obtaining h igh expression levels. The results also suggest the importance of the proli ne 50 residue in the recovery yields of human pancreatic ribonuclease, All the variants produced are pure and homogeneous. Their homogeneity has been demonstrated by cation-exchange and reversed-phase chromatography and by ma ss spectrometry analysis. Moreover, enhancement of human pancreatic ribonuc lease thermal stability is observed when residues R4, K6, Q9, D16, and S17 are changed to the corresponding residues of bovine seminal ribonuclease (C ) 1999 Academic Press.