Production Of Horseradish Peroxidase Research Articles

Protein Ligation-Assisted Reconstitution of Split HRP Fragments for Facile Production of HRP Fusion Proteins in E. coli.

Horseradish peroxidase (HRP) is a pivotal biocatalyst for biosensor development and fine chemical synthesis. HRP proteins are mostly extracted and purified from the roots of horseradish because the solubility and productivity of recombinant HRP in bacteria are significantly low. In this study, we investigate the reconstitution system of split HRP fragments to improve its soluble expression levels in E. coli allowing the cost-effective production of bioactive HRPs. To promote the effective association between two HRP fragments (HRPn and HRPc), we exploit SpyTag-SpyCatcher chemistry, a versatile protein coupling method with high affinity and selectivity. Each HRP fragment was genetically fused with SpyTag and SpyCatcher, respectively, exhibiting soluble expression in the E. coli cytoplasm. The engineered split HRPs were effectively and irreversibly reconstituted into a biologically active and stable assembly that can catalyze intrinsic enzymatic reactions. Compared to the chaperone co-expression system, our approach shows that the production yield of soluble HRP is comparable, but the purity of the final product is relatively high. Therefore, our results can be applied to the high-yield production of recombinant HRP variants and other difficult-to-express proteins in bacteria without complex downstream processes.

Stepwise Optimization of Inducible Expression System for the Functional Secretion of Horseradish Peroxidase in Saccharomyces cerevisiae.

Horseradish peroxidase (HRP) is a plant-derived glycoprotein that can be developed as a food additive to cross-link proteins or biopolymers. Although Saccharomyces cerevisiae has advantages in the production of food-grade HRP, the low expressional level and inefficient secretion hindered its application values. After comparing the effects of constitutive and inducible expression on cell growth, the strength of HRP expression was roughly tuned by replacing core regions of the promoter in the GAL80-knockout strain and further finely tuned by terminator screening. Additionally, the most suitable signal peptide was selected, and the pre-peptide with pro-peptides was modified to balance the transport of HRP in the endoplasmic reticulum. The extracellular HRP activity of the best strain reached 13 506 U/L at the fermenter level, 330-fold higher than the previous result of 41 U/L in S. cerevisiae. The strategy can be applied to alleviate the inhibition of cell growth caused by the expression of toxic proteins and improve their secretion.

Production of Recombinant Horseradish Peroxidase in an Engineered Cell-free Protein Synthesis System.

One of the main advantages of a cell-free synthesis system is that the synthetic machinery of cells can be modularized and re-assembled for desired purposes. In this study, we attempted to combine the translational activity of Escherichia coli extract with a heme synthesis pathway for the functional production of horseradish peroxidase (HRP). We first optimized the reaction conditions and the sequence of template DNA to enhance protein expression and folding. The reaction mixture was then supplemented with 5-aminolevulinic acid synthase to facilitate co-synthesis of the heme prosthetic group from glucose. Combining the different synthetic modules required for protein synthesis and cofactor generation led to successful production of functional HRP in a cell-free synthesis system.

Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies.

Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.

Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis.

Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.

Soluble expression of horseradish peroxidase in Escherichia coli and its facile activation

Horseradish peroxidase (HRP) is widely used as a marker enzyme in immunoassays and biosensors, and can possibly be used in industries such as waste water treatments or fine chemical synthesis. Cost-effective production of active HRP is thus very important in the related fields. Also, engineering of HRP for its better performance in the designated application is expected to make the enzyme even more important in several areas of research and industry. One of obstacles to this end and to the large scale production of the enzyme has been its facile expression in a bacterial host. Here we show that HRP could be overexpressed as a soluble form by fusing the enzyme with Escherichia coli phosphoglycerate kinase (PGK). After simple incubation with calcium ion, hemin, and oxidized glutathione, PGK-HRP could be fully activated showing a higher molar specific activity than plant-derived HRP. Our established procedure did not use tedious and inefficient refolding steps that have been used to activate HRP produced as inclusion bodies and thus is superior in its overall yield (>72mg purified HRP conjugate per L culture) to existing methods. By co-expressing PGK-HRP with ferrochelatase in a special host that permitted the formation of disulfide bonds in the cytoplasm, the activation steps could be simplified even though the resulting specific activity was low.

A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli

Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E. coli and investigated and compared both strategies, (a) the production of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the production of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titre of 100mgL−1 cultivation broth, and were able to produce 48mg active HRP per litre cultivation broth in the periplasm. In terms of biochemical properties, soluble HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and production. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amounts, which are then refolded.

Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris

Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible PAOX1 promoter, the strong constitutive PGAP promoter, and a novel bidirectional promoter PHTX1. Ultimately, coproduction of HRP and active Hac1 under PHTX1 control yielded a recombinant HRP titer of 132mg/L after 56h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme.

Effects of different media supplements on the production of an active recombinant plant peroxidase in a Pichia pastoris Δoch1 strain

Recombinant protein production in microorganisms is one of the most studied areas of research in biotechnology today. In this respect the yeast Pichia pastoris is an important microbial production host due to its capability of secreting the target protein and performing posttranslational modifications. In a recent study, we described the development of a robust bioprocess for a glyco-engineered recombinant P. pastoris strain where the native α-1,6-mannosyltransfrease OCH1 was knocked out (Δoch1 strain). This strain produced the glycosylated enzyme horseradish peroxidase (HRP) with more homogeneous and shorter surface glycans than the respective benchmark strain. However, the recombinant Δoch1 strain was physiologically impaired and thus hard to cultivate. We faced cell cluster formation, cell lysis and consequent intensive foam formation. Thus, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on (1) cell physiology, (2) cell morphology, (3) cell lysis, (4) productivity and (5) product purity in a multivariate manner. However, not only process parameters might influence these characteristics, but also media supplements might have an impact. Here, we describe the effects of different heme-precursors as well as of a protease-inhibitor cocktail on the production of active HRP in therecombinant P. pastoris Δoch1strain.

Enzyme Linked Immuno Mass Spectrometric Assay (ELIMSA)

A new technology termed ELIMSA combines the specificity and enzymatic amplification of Enzyme Linked Immunosorbent assay (ELISA) with the sensitivity and flexibility of mass spectrometry (MS). At present, substrates for the reporter enzymes horseradish peroxidase (HRP) or alkaline phosphatase (AP) yield colored, fluorescent or luminescent products. The central concept of ELIMSA is that the reporter enzymes HRP and AP yield products that ionize efficiently with a high signal to noise ratio that can be measured by mass spectrometry. The reporter enzymes HRP or AP may be covalently attached to a specific detection probe such as a protein or an antibody to bind their target analyte and then catalyze the rapid production of ionizable, small-molecules. The use of mass spectrometry to measure small molecule products may commonly reach femto to picomol amounts on the column with high signal to noise ratio. Mass spectrometry combined with the enzyme amplification in ELISA provides absolute sensitivity to detect attomol of PSA and was comparable to, or more sensitive, than radio immune assays and electrochemical detectors but with only existing reagents and equipment. ELIMSA permits monitoring of multiple substrates and products and provides comparison to absolute standards. There is an urgent need to detect and quantify low abundance proteins such as hormones, chemokines, cytokines, and others that exist at attomolar concentrations under physiological conditions by ELIMSA. A sensitive method for the quantification of immunological assays is obtained by applying mass spectrometry to detect the products of the alkaline phosphatase (AP) and horseradish peroxidase (HRP) enzyme reactions. There are many molecules from human subjects or micro-organisms that are of great importance to medicine, industry, nutrition or the environment that need to be repeatedly analyzed and are often near to, or beyond, the edge of existing analytical technology. The presence of molecules in biological samples, industrial products or the environment may be detected by probes that bind to the target analyte. Combining the reporter enzymes from ELISA with sensitive liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) will permit the sensitive detection and quantification of the molecular probes by Enzyme Linked Immuno Mass Spectrometric Assay (ELIMSA). The flexibility and sensitivity of mass spectrometry to measure large numbers of compounds simultaneously should permit the quantification of multiple ELIMSA reactions at separate mass-to-charge (m/z) ratios. Hence ELIMSA and it variants should permit the rapid and simple detection and quantification of many molecules over the complete range of biologically important concentrations without the use of radiolabels using only existing antibodies, reagents and instruments. Antibodies coupled to reporter enzymes that are widely used in biomedical and environmental applications can now be detected and quantified using ultra sensitive mass spectrometry to create a sensitive and flexible ELIMSA system. Absolute standards of analytes or enzyme product may serve as a reference.

Purification of a recombinant plant peroxidase produced in Pichia pastoris by a simple 2-step strategy

The enzyme horseradish peroxidase (HRP), which is frequently applied in industry and medicine, is primarily isolated from plant. This purification procedure is costly and the obtainable amount of HRP from the horseradish root is low. However, recombinant HRP (rHRP) produced in yeast is hyperglycosylated rendering the subsequent purification cumbersome and the recombinant production of HRP in yeast not competitive.In this study, we screened different common techniques to develop a fast and efficient purification strategy for hyperglycosylated rHRP expressed in Pichia pastoris. We demonstrated that the extensive glycosylation pattern on the surface of rHRP masked its physico-chemical properties, which is why standard purification strategies were rather unsuccessful. Only switching the strategies to a flowthrough mode gave satisfactory results. After determining the optimal operation conditions in a multivariate Design of Experiments approach, we present a simple 2-step strategy for the purification of hyperglycosylated rHRP. Combining a hydrophobic charge induction chromatography operated in flowthrough mode and a size-exclusion chromatography, we were able to purify rHRP more than 12-fold from a specific activity of 80U/mg to more than 1000U/mg.

Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method

Fil: Targovnik, Alexandra Marisa. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Departamento de Microbiologia, Inmunologia y Biotecnologia. Catedra de Microbiologia Industrial y Biotecnologia; Argentina

Heat Stress Increases Protein Antigen Transport Across the Intestinal Epithelium Via a Mechanism of Impairing Proteolytic Enzymatic Activity

It has not been fully understood how intact protein antigens escape digestion in the course of absorption. The present study was designed to investigate the mechanism that heat stress induced an increase in intact protein antigen absorption. Human colonic cell line Caco-2 cells were treated with high temperature (37 to 43 degrees C) for 60 min. Epithelial permeability was evaluated by horseradish peroxidase (HRP) flux and dextran flux. Activity of the major intracellular proteolytic enzyme, acid phosphatase, in Caco-2 cells was determined. HRP products in Caco-2 cells were observed by electron microscopy (EM) and analyzed with a computerized image processing system. Heat stress significantly increased intact protein HRP transport across Caco-2 monolayers, decreased acid phosphatase activity of the cells, and significantly reduced transepithelial electric resistance of Caco-2 cells. EM results showed that HRP transport across Caco-2 monolayers occurred mainly via the intracellular pathway.

Methylene blue and azure B oxidation by horseradish peroxidase: a comparative evaluation of class II and class III peroxidases

We have previously shown that the oxidation of methylene blue (MB) or azure B (AB) by Phanerochaete chrysosporium lignin peroxidase (LiP) (class II) occurs via stepwise N-demethylations followed by aromatic ring cleavage under selective reaction conditions related to methylene blue:H2O2 or azure B:H2O2 stoichiometry. In this work, we compare the oxidation of the same dyes by horseradish peroxidase (HRP) of plant origin (class III) and compare LiP and HRP reactions and products. Results show HRP is able to N-demethylate both dyes, but exhibits much slower reaction kinetics than LiP and requires higher H2O2 concentrations. Product yields are also different for HRP, and contrary to LiP, HRP is unable to achieve aromatic ring cleavage. Azure C (AC), which is formed by sequential oxidation of either MB or AB, was a major reaction product in HRP-mediated reactions. Yields of AC up to 60% were obtained, suggesting a potential enzymatic route for AC production that compares quite favorably to the chemical route yield of 35%.

Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli.

Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 μM isopropyl-β- d-thiogalactopyranoside (IPTG) at 37°C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.

Improvement of Productivity of Active Horseradish Peroxidase in Escherichia coli by Coexpression of Dsb Proteins.

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 microM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.

Endocytosis of azurophil granules released from neutrophils by junctional epithelial cells of rat gingiva.

A cytochemical study using diaminobenzidine (DAB) reaction and horseradish peroxidase (HRP) tracer or DAB reaction alone was performed on junctional epithelium (JE) of young (32-to 42-day-old) and old (100-day-old) rats to investigate how JE cells endocytose the azurophil granules released from neutrophils, as JE cells do with HRP.HRP was endocytosed by JE cells, and the endocytosed HRP products fused with one another or cytoplasmic vacuoles and formed phagolysosomes of amorphous shape, which digestion of the HRP seemed to be progressive. It was thus certified that JE cells possess endocytotic ability. In old rat gingivae incubated with only DAB medium, neutrophils were found in the JE and contained endogenous peroxidasepositive round, ovoid and elongated azurophil granules. In particular, at the coronal portion of JE, endogenous peroxidase-positive round ovoid and elongated reaction products were numerously detected in the intercellular spaces of the JE cells and within the JE cells. Judging from the cytochemical and morphological resemblances between the endogenous peroxidase-positive reaction products and the azurophil granules in the neutrophils, the reaction products in the intercellular spaces corresponded to the azurophil granules released from neutrophils, and those in the JE cells corresponded to the azurophil granules endocytosed by the JE cells. Furthermore, the endogenous peroxidase-positive reaction products endo cytosed by JE cells also formed the same amorphous phagolysosomes as formed by HRP. It was, therefore, concluded that, at the coronal portion of the JE of old rat gingivae, the JE cells may endocytose and digest lysosomes (azurophil granules) released from neutrophils to protect the gingival tissue.

Ultrastructural identification of synaptic terminals from cortical axons and from collateral axons of geniculo-cortical relay cells in the perigeniculate nucleus of the cat.

Electron microscopic analysis of sections of the perigeniculate nucleus (PGN) of the cat processed with horseradish peroxidase (HRP) histochemistry after massive injections of this enzyme in the visual cortex showed two types of synaptic terminals labeled with HRP reaction products. One type (RLD terminals) is characterized by round synaptic vesicles, large size, dark mitochondria and asymmetrical synaptic contacts with somata and dendrites. The second type (RSD terminals) is characterized by round synaptic vesicles, small size, dark mitochondria and asymmetrical synaptic contacts with dendrites. The HRP+ RSD terminals, which were also found in the dorsal lateral geniculate nucleus (LGN), are interpreted as terminals of cortical origin both in the PGN and LGN, since previous studies have identified cortical terminals as being of RSD type in the LGN and in other thalamic nuclei. The HRP+ RLD terminals are interpreted as synaptic terminals of collaterals axons of geniculocortical relay cells in the PGN labeled by retrograde transport of HRP from the cortex. In addition, in semithin and ultrathin sections somata in the PGN were never found labeled with HRP products indicating the absence of a PGN projection to the visual cortex.