Recombinant HRP

The successful expression of recombinant plant peroxidases such as HRP C has been a major focus of research in a number of laboratories. Three synthetic HRP C genes based on the amino acid sequence determined by Welinder (36, 47) were synthesized independently in order to initiate this work (63-65). A number of different expression systems have been evaluated (64, 66-73), a summary of which is presented in Table I. Refolding of recombinant HRP C isolated from inclusion... 

Aspergillus oryzae Various fusions between the S3mthetic HRP C gene and CIP at both N- and C-termini. Low Recombinant HRP C detected on Western blots (66). 

The Ki values determined for wild-type plant enzymes are given at the head of the table, followed by data obtained for wUd-type recombinant HRP C and site-directed mutants of HRP C. Solution conditions typically 10-50 mM sodium phosphate or 10 mM sodium MOPS, pH 7.0, 25°C. 

Scheme 3. Structures of hemin, the iron(III) complex of protoporphyrin IX, and recombinant HRP isoenzyme C (29).

A 3-A structure has been published for isoenzyme E5 of HRP (33), but no high-resolution X-ray structure is yet available for a plant peroxidase. It is anticipated that over the next few years structural data will be available for a number of plant peroxidases. Crystallographic studies on recombinant HRP isoenzyme c (HRP-C) (34), barley peroxi-... 

Figure 3 Calibration plots for glucose in the presence (O) and in the absence ( ) of 0.1 mM acetaminophen. The calibrations were recorded with graphite electrodes modified with bienzyme layer of (A) native HRP and glucose oxidase or (B) recombinant HRP and gln-cose oxidase. The measurements were done in a flow-injection mode, with a flow carrier of 20 mM phosphate buffer, pH 7 containing 0.1 M KCl. Applied potential was -50 mV vs. Ag/AgCl/0.1 M KCl.

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