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 a-factor pre-pro segment) or Escherichia coli (fused to the pelB signal sequence). HP-RNase produced in S. cerevisiae was secreted into the medium as an active enzyme, isolable at 0.1-0.2 mg/liter of culture. 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 fraction of the cell lysate. Renaturation of the reduced and denatured protein produced active, homogeneous enzyme recoverable at 1 mg/liter of culture. The N terminus of the HP-RNase produced from the bacterial expression system was processed fully in vivo. The yeast system, combined with techniques that allow detection of picograms of ribonuclease activity, offers a sensitive probe for studies of post-translational modification and secretory targeting in eukaryotic cells. The bacterial system enables studies both to reveal new structure-function relationships in ribonucleases and to evaluate the use of HP-RNase as a cytotoxin that is tolerated by the human immune system.
Ribó
M
M
Unitat de Bioquímica i Biologia Molecular, Departament de Biologia, Universitat de Girona, Girona, Spain.
delCardayré
S B
SB
Raines
R T
RT
de Llorens
R
R
Cuchillo
C M
CM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003597:Cytosine Nucleotides / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D011066:Poly C / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D003597:Cytosine Nucleotides; D009841:Oligonucleotides; D011994:Recombinant Proteins; D011066:Poly C; C019310:cifostodine; D012259:Ribonuclease, Pancreatic