Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.
For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.
Taylor
M A
MA
AFRC, Institute of Food Research, Reading, UK.
Pratt
K A
KA
Revell
D F
DF
Baker
K C
KC
Sumner
I G
IG
Goodenough
P W
PW
D000595:Amino Acid Sequence; D001483:Base Sequence; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D011487:Protein Conformation; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry*; D012995:Solubility
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D010206:Papain