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Title
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Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme.
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Authors
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A.T.Smith,
N.Santama,
S.Dacey,
M.Edwards,
R.C.Bray,
R.N.Thorneley,
J.F.Burke.
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Ref.
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J Biol Chem, 1990,
265,
13335-13343.
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PubMed id
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Abstract
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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.
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