Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme.
A synthetic gene encoding horseradish peroxidase isoenzyme C (HRP C) has been synthesized and expressed in Escherichia coli. The nonglycosylated recombinant enzyme (HRP C*) was produced in inclusion bodies in an insoluble inactive form containing only traces of heme. HRP C* was solubilized and conditions under which it folded to give active enzyme were determined. Folding was shown to be critically dependent upon the concentrations of urea, Ca2+, and heme and on oxidation by oxidized glutathione. Purification of active HRP C* from the folding mixture gave a peroxidase, with about half the activity of HRP C. Glycosylation is thus not essential for correct folding and activity. The C-terminal and N-terminal extensions to HRP identified previously in cloned cDNA sequences are also not required for correct folding. However, Ca2+ appears to play a key role in folding to give the active enzyme. The overall yield of purified active enzyme was 2-3%, but this could be increased by reprocessing material that precipitated during folding.
Biochemistry Laboratory, University of Sussex, Brighton, United Kingdom.
A T
AT
Smith
N
N
Santama
S
S
Dacey
M
M
Edwards
R C
RC
Bray
R N
RN
Thorneley
J F
JF
Burke
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000302:isolation & purification; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005813:Genes, Synthetic; D006418:Heme / Q000494:pharmacology*; D006735:Horseradish Peroxidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry; D014508:Urea / Q000494:pharmacology
D001051:Apoenzymes; D011994:Recombinant Proteins; D006418:Heme; D014508:Urea; D006735:Horseradish Peroxidase; D010544:Peroxidases; D002118:Calcium