PRODUCTION OF HUMAN PANCREATIC RIBONUCLEASE IN SACCHAROMYCES-CEREVISIAE AND ESCHERICHIA-COLI

Human pancreatic ribonuclease (HP-RNase) has considerable promise as a therapeutic agent, Structure-function analyses of HP-RNase have been impeded by the difficulty of obtaining the enzyme from its host. Here, a gene encoding HP-RNase was designed, synthesized, and inserted into two expression vectors that then direct the production of HP-RNase in Saccharomyces cerevisiae (fused to either an unmodified or a modified alpha-factor pre-pro segment) or Escherichia coil (fused to the pelB signal sequence), RP-RNase produced in S. cerevisiae was secreted into the medium as an active enzyme, isolable at 0.1-0.2 mg/liter of cultu re. This isolate was heterogeneous due to extensive glycosylation and incomplete maturation of the pre-pro segment, HP-RNase produced in E. coli with the pET expression system was purified from the insoluble fr action of the cell lysate, Renaturation of the reduced and denatured p rotein produced active, homogeneous enzyme recoverable at 1 mg/liter o f culture, The N terminus of the HP-RNase produced from the bacterial expression system was processed fully in vivo. The yeast system, combi ned with techniques that allow detection of picograms of ribonuclease activity, offers a sensitive probe for studies of post-translational m odification and secretory targeting in eukaryotic cells, The bacterial system enables studies both to reveal new structure-function relation ships in ribonucleases and to evaluate the use of HP-RNase as a cytoto xin that is tolerated by the human immune system. (C) 1996 Academic Pr ess, Inc.