ISSN 00062979, Biochemistry (Moscow), 2015, Vol. 80, No. 4, pp. 408416. © Pleiades Publishing, Ltd., 2015. Original Russian Text © V. G. Grigorenko, I. P. Andreeva, M. Yu. Rubtsova, A. M. Egorov, 2015, published in Biokhimiya, 2015, Vol. 80, No. 4, pp. 480488.


Recombinant Horseradish Peroxidase: Production and Analytical Applications V. G. Grigorenko1*, I. P. Andreeva1, M. Yu. Rubtsova1, and A. M. Egorov1,2 1

Lomonosov Moscow State University, Chemical Faculty, 119991 Moscow, Russia; fax: +7 (495) 9392742; Email: [email protected] 2 Russian Medical Academy of PostGraduate Education, Microbiology Department, 125993 Moscow, Russia Received November 14, 2014 Revision received December 9, 2014 Abstract—Horseradish peroxidase is a key enzyme in bio and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli cells and the subsequent refolding of the resulting protein yield a recombinant enzyme that is comparable in its spectral and catalytic characteristics to the native plant peroxidase. Genetic engineering approaches allow production of recombinant peroxidase conjugates with both protein antigens and Fab antibody fragments. The present article reviews the use of recombinant horseradish peroxidase as the marker enzyme in ELISA procedures as well as in amperometric sensors based on direct electron transfer. DOI: 10.1134/S0006297915040033 Key words: horseradish peroxidase, recombinant conjugate, amperometric biosensor

Isoenzyme C of horseradish peroxidase (HRP, EC is a ubiquitous plant heme and Ca2+contain ing enzyme that belongs to the family of plant peroxidas es [13] catalyzing oneelectron oxidation of various organic and inorganic substrates by hydrogen peroxide. Plant peroxidases are used in different biotechnological processes, such as biobleaching, utilization of organic compounds (phenols, dyes, lignin) being formed during a number of technological and other processes, while horseradish peroxidase is widely used in analytical bio chemistry and biotechnology as the marker enzyme for immunochemical detection of antibodies, DNA, and low molecular weight compounds. Due to wide substrate specificity and high catalytic activity and stability, HRP is often employed in bio and immunosensors, chemilumi nescent, fluorescent, and electrochemical detection sys tems, as well as in DNA and protein microchips with colorimetric detection [48]. Successes in the heterological expression of the per oxidase gene and determination of the crystal structure of HRP opened new opportunities for the investigation of structural–functional relationships by means of genetic Abbreviations: FABP, fatty acidbinding protein; HRP, horse radish peroxidase; LO, Llysineαoxidase; rHRP, recombi nant horseradish peroxidase. * To whom correspondence should be addressed.

engineering approaches. The main problem of HRP expression is the complex structure of the enzyme: it is a glycoprotein containing a heme in its active site. There are several ways of expression of the HRP gene. For example, expression in the baculovirus system resulted in the production of the enzyme in a soluble active and gly cosylated form [9]. However, this system was not widely used, being rather timeconsuming, complex, and expen sive compared to expression in E. coli cells. Expression of HRP in yeast resulted in the production of the enzyme in a soluble and active form, but with low yield [10]. For the production of recombinant wildtype HRP (rHRP), as well as its mutant forms, the expression system of E. coli cells is commonly used [11, 12]. Recombinant HRP is synthesized in the cytoplasm of E. coli cells within inclusion bodies in an insoluble and inactive form con taining trace amounts of the prosthetic group. The process of the refolding and reactivation of the apoperoxidase in the presence of the heme prosthetic group is a complex multistep process, since the protein molecule contains four disulfide bonds and two Ca2+ ions. Besides, the plant enzyme contains eight glycosylation sites (18% carbohy drates by mass). Previously, the crystal structure of rHRP was determined, and the protein molecule was shown to be composed of two domains containing 10 αhelices [13]. One of the factors leading to the formation of inclu sion bodies in the case of heterological expression of


RECOMBINANT HORSERADISH PEROXIDASE recombinant proteins with disulfide bonds in E. coli cells is an active system maintaining high reducing potential of the cytoplasm, which prevents the correct disulfide bond ing and formation of the native structure of such proteins [14]. An alternative approach developed for the produc tion of the recombinant protein in soluble and active form was its expression in the bacterial periplasm, where the oxidative potential promotes the formation of stable disulfide bonds. For this purpose, an expression vector encoding HRP and a signal peptide at the Nterminus was created. After the translocation, the signal peptide is removed, yielding the correctly folded protein with disul fide bonds. This approach was successfully used for the production of soluble active recombinant rHRP, but with rather low yield (~0.5 mg protein per liter of E. coli cell culture) [15]. For analytical application of rHRP, for example in biosensors, more efficient and technological convenient method was required for the production of the recombi nant enzyme. The insertion of the hexahistidine sequence (6× His) into the Cterminal region of HRP made it pos sible to isolate the enzyme using metalchelate affinity chromatography, which significantly facilitated the reac tivation process and purification of rHRP from the inclu sion bodies. Besides, the yield of the recombinant prepa ration increased 3fold, particularly due to the efficient isolation of rHRP from diluted refolding solutions [15]. Due to relatively high yield (~10 mg per liter of the cell culture) and high specific activity of the recombinant enzyme, obtained by the described refolding procedure, the rHRP preparation can be used for development of biosensors based on direct electron transfer from an elec trode to immobilized rHRP [4, 6] and for enhancing sen sitivity of sensors based on the reaction of enhanced chemiluminescence [7].

BIOSENSORS BASED ON RECOMBINANT HORSERADISH PEROXIDASE Peroxidase immobilized on an electrode and capable of catalyzing direct electron transfer allows creation of a mediatorless biosensor for determination of peroxides and organic compounds producing peroxides during the oxidation by corresponding oxidases (glucose oxidase, lysine oxidase, etc.) However, in the absence of a media tor, the efficiency of direct electron transfer in the gold electrode–HRP system is very low [4, 5]. The most prob able reason for this is a large distance between the elec trode surface and the active site of HRP due to the glyco sylation of the plant enzyme and/or incorrect orientation of the enzyme molecule on the electrode surface. Besides, one of the most serious problems is the strength of the adsorption of the protein molecule on the electrode sur face. The method of covalent immobilization by chemical linking increases the distance between the active site of BIOCHEMISTRY (Moscow) Vol. 80 No. 4 2015


the enzyme and the electrode, hampering direct electron transfer. Known approaches for immobilization of bio molecules allowing electron transfer consist in firm chemisorption of a number of functional groups on the gold surface [4, 5]. The gene engineering techniques allow construction of rHRPs with changed molecule surface by introducing amino acid residues (cysteine, histidine) containing suit able functional groups for the oriented immobilization of the enzyme [15]. Gold electrodes modified with recombi nant forms of HRP demonstrated high and stable amperometric response in the reaction of biocatalytic reduction of hydrogen peroxide due to direct electron transfer between the HRP and the electrode, so this approach seemed to be useful for the elaboration of non mediator biosensors [6]. Combination of rHRP with Llysineαoxidase (LO) that exhibits high catalytic activity in reducing hydrogen peroxide was used to create a mediatorless bien zyme biosensor for Llysine that was capable of working at moderate potential values (0 to –50 mV) in the case of direct electron transfer between the electrode surface and the active site of the enzyme (Fig. 1). In this case, the concentration of Llysine is proportional to the concen tration of hydrogen peroxide that is released during its enzymatic oxidation. An amperometric bienzyme biosen sor for determination of Llysine was developed based on LO (class of oxidoreductases, EC from Trichoderma viride and rHRP with a histidine tag (6× His) on the Cterminus. The two enzymes were coimmobi lized on the surface of gold polycrystalline electrodes. The efficient direct electron transfer between the surface of the gold electrode and the immobilized rHRP allowed mediatorless determination of hydrogen peroxide that was produced during the enzymatic oxidation of Llysine in stoppedflow mode (Fig. 2). The minimal concentra tion of Llysine for the described method was 1 μM, and the sensitivity of the determination was 0.03 A·cm–2·M–1 at –50 mV relative to the silverchloride reference elec trode [46].

RECOMBINANT CONJUGATES IN ANALYSIS Horseradish peroxidase is one of the most used marker enzymes in immunodiagnostics. The highly sensi tive methods of enzymelinked immunosorbent assay (ELISA) require conjugates of marker enzymes with anti gens or antibodies. However, all chemical methods of preparation of the conjugates usually result in a partial inactivation of the enzymes and in heterogeneity of the conjugates, which affects the reproducibility, specificity, and sensitivity of the analysis. The gene engineering tech niques allow creation of recombinant conjugates with protein antigens and antibodies containing structural parts of both a marker enzyme and an antigen/antibody.





Lysine oxidase

Electrode Fig. 1. Bienzyme amperometric sensor based on horseradish peroxidase and lysine oxidase.

Such fusion proteins have some advantages compared to conjugates obtained by chemical methods. Namely, they are homogeneous by composition, exhibit stoichiometry of 1 : 1; they are reproducible and exhibit functional activity of both the marker protein and the antigen/anti body [16, 17]. Recombinant fusion antigens were obtained with the bacterial enzymes βgalactosidase [1820] and alkaline

1 j, μA/cm2



0.01 0.1 1 log[Substrate], μM

2.0 j, μA/cm2

2 0 10


1.5 1.0 2 0.5








[Substrate], μM

Fig. 2. Dependence of steadystate current density on concentra tion of Llysine (1) and H2O2 (2) in a flowinjection system for gold electrodes modified with histidinetagged rHRP together with LO. The electrode potential was –50 mV relative to Ag/AgCl in 0.1 M KCl, flow rate of the substrate in PBS was 900 μl/min, pH 6.0. Curve 1 was obtained in the stoppedflow mode.

phosphatase [2123] that were expressed in soluble form in E. coli cells, as well as with bioluminescent and fluo rescent marker proteins such as aequorin from Aequorea victoria and green fluorescent protein [24, 25]. Recombinant conjugates of protein A with enzymes, par ticularly with βlactamase [26] and luciferase [27] were obtained. Besides, geneengineering techniques are espe cially profitable for small peptides with numerous func tional groups that are difficult to detect [28] and for human proteins that are often difficult to obtain in large amounts [29]. Recombinant antibodies fused with differ ent enzymes (alkaline phosphatase [3034], luciferase [35], and peroxidase from Arthromyces ramosus [36]) were also obtained. While βgalactosidase is expressed in soluble form in the cytoplasm, disulfidecontaining alkaline phosphatase is secreted into the periplasm [37, 38]. A disadvantage of the bacterial alkaline phosphatase is lower specific activi ty compared to that of the alkaline phosphatase from calf pancreas that is used for preparation of conjugates by chemical linking. This problem can be partially solved by using more active, genetically engineered mutants of the bacterial enzyme [39]. The main problem concerned with βgalactosidase and alkaline phosphatase conjugates is the oligomeric structure of these enzymes (tetrameric and dimeric, respectively), this increasing significantly the affinity of the conjugates compared to the free antigen. This is an undesirable effect, especially when using com petitive schemes of enzyme immunoassay. On the other hand, HRP that is used extensively for preparation of the conjugates [20] can be expressed in E. coli cells only within inclusion bodies [11, 12], which pre vented the application of the recombinant enzyme for a long time. Successes in heterologous expression of the HRP gene in E. coli cells and in the reactivation of the BIOCHEMISTRY (Moscow) Vol. 80 No. 4 2015





Fig. 3. Scheme of cloning (a) and the spatial model (b) of HRP–FABP fusion protein.

recombinant HRP containing an oligohistidine sequence on the Cterminus [15] enabled production of recombi nant fusion proteins with peroxidase as the marker enzyme and their application in the enzyme immunoas say.

RECOMBINANT CONJUGATES OF PROTEIN ANTIGENS WITH HRP Recombinant fusion proteins with HRP were first obtained with the protein antigen FABP (fatty acidbind ing protein) and employed for the detection of this pro tein by competitive enzyme immunoassay [40]. FABP is a small (~15 kDa) cytosolic protein that is belongs to the family of intracellular lipidbinding proteins involved in metabolism of fatty acids. Cardiactype FABP is present in myocardium in large amounts (0.56 mg/g cardiac tis sue) and differs from other types of FABP in immunolog ical characteristics. Due to a small size, the protein is eas ily released from damaged cardiomyocytes into the bloodstream. This specific feature, as well as the tissue specificity compared to myoglobin, make FABP a prom ising marker for early diagnostics of the acute myocardial infarction [29, 41]. To obtain peroxidase fused with FABP (HRP– FABP), the expression vector pETHRPFABPhis was used (Fig. 3), in which the DNA region encoding FABP was cloned into the XmaIII site in the corresponding reading frame after HRP codons and before 6× His [15]. The fusion protein was produced in the cytoplasm of E. BIOCHEMISTRY (Moscow) Vol. 80 No. 4 2015

coli cells in inclusion bodies. The protein was refolded and reactivated according to the procedure elaborated earlier for recombinant HRP containing 6× His at the C terminus [15]. The HRP–FABP fusion protein exhibited maximal absorption at 403 nm, this corresponding to the maxi mum of the Soret band, as in the case of the recombinant and native HRP, which indicated the unchanged microenvironment of Fe3+ (coordination with distal and proximal histidines of the heme). These data suggested that the Cterminal “extension” of HRP with 15kDa FABP did not affect the catalytic activity of the fusion protein, which corresponded to the values of the specif ic activity of plant HRP and recombinant histagged HRP (~1000 U/mg with substrate ABTS) [13]. At the same time, sandwich ELISA showed that the fusion pro tein interacted with both monoclonal and polyclonal antibodies specific to FABP, which indicated the integri ty of the antigen determinants of the FABP molecule [29, 40]. To detect FABP, the competitive ELISA procedure was employed, which takes less time compared to sand wich immunoassays. The method is based on the compe tition of FABP from a test sample and the FABP fused with HRP for the binding sites of the antibodies against FABP immobilized on the surface of a polystyrene plate wells. For comparison, a preparation of conjugates of plant HRP with FABP was synthetized using periodate oxidation of oligosaccharides [42]. Typical calibration curves (Fig. 4) demonstrate a wide range of FABP con centration detected by the method (1.5500 ng/ml) and a


GRIGORENKO et al. creation of a recombinant HRP fused with human myo globin, another important marker for the early diagnos tics of acute myocardium infarction.



0.6 0.4 2 0.2


0 1



FABP concentration, ng/ml

Fig. 4. Calibration curves for detection of FABP: 1) recombinant HRP–FABP fusion protein; 2) HRP–FABP conjugate obtained by chemical method.

low detection limit (1.5 ng/ml or 0.05 ng per well). The variation coefficient did not exceed 8% [40]. These results agree with the literature data for sandwich ELISA meth ods with the use of monoclonal antibodies (0.020.1 ng per well) [29, 41]. At higher concentrations, FABP better competes with the recombinant HRP–FABP fusion pro tein than with the chemically prepared conjugate for the binding with polyclonal antibodies. This could be explained by the presence of an HRP molecule bound with several FABP molecules in the chemically obtained conjugates, which increase the affinity due to the multiple binding with the immobilized antibodies. Thus, the recombinant HRP–FABP fusion protein that exhibits homogenous composition, stoichiometry of 1 : 1, and specific activity exceeding 2fold the activity of the syn thetic conjugate has an advantage while using the com petitive ELISA procedure. To test the elaborated method, samples of the blood serum of a patient with acute myocardial infarction were analyzed within 24 h after hospitalization (Fig. 5) [40]. The values of FABP concentration obtained by the com petitive ELISA procedure correlated well with data obtained in the framework of the EUROCARDI project using the sandwich ELISA procedure and electrochemi cal immunosensor assay [29, 41]. Thus, for the first time the possibility of reproducible production of recombinant conjugate of the protein anti gen with HRP as the marker enzyme and its application in competitive ELISA for detection of the clinically important analyte FABP was demonstrated [40]. This approach offers new opportunities for the application of the commonly used marker enzyme HRP in the compet itive ELISA procedure with the use of recombinant fusion proteins. For example, a concept was developed for the

Functional expression of the recombinant HRP fused with antibody fragments in E. coli cells is associated with a number of difficulties. In E. coli cells, the system of posttranslational glycosylation is absent, which leads to low solubility and aggregation of the produced protein. This problem could be solved by changing the expression system. For example, it was shown that the methy lotrophic yeast Pichia pastoris is more suitable for the expression of the antibodies than E. coli [43, 44]. Individually, HRP [45] and antibody fragments [46] (both the singlechain scFv [47, 48] and Fab [49] forms) were successfully expressed in P. pastoris cells. Moreover, using this expression system, scFv fragments fused with the IgGbinding domain of protein A, with biotinlike peptide sequence, and with Fcfragments of IgG1 were produced [5052]. Besides, the expression in P. pastoris in the secreted form was shown to simplify significantly the biotechnological application of the process [53]. Success in functional expression of HRP and anti bodies in the secreted form in P. pastoris [45, 54] opened the way for creation of HRP fused with antibodies for application in enzyme immunoassays. However, creation of recombinant fusion proteins is a rather complex and nontrivial task, since it is impossible to predict reliably the structure of the produced fusion protein. Therefore, the functional activity of both the marker enzyme and the antigen can be lost because of incorrect folding of the two components of the chimeric protein.

FABP concentration, ng/ml



1000 1 800


600 400 200

3 0






Time, h

Fig. 5. Comparison of quantitative assays for FABP in human blood serum (profile of patient No. 14): 1) sandwich ELISA; 2) competitive ELISA; 3) electrochemical immunosensor assay.

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Fig. 6. Cloning schemes and spatial models of Fab–HRP and HRP–Fab recombinant fusion proteins (left and right panels, respectively).

The expression system for the production of recom binant HRP fused with antibody Fab fragments was developed using a pPICZαB vector and P. pastoris X33 cells (Invitrogen, USA). The expression is induced by methanol and results in the production into the culture medium of two types of fusion proteins, in which the per oxidase part is linked by a short flexible sequence (Gly4Ser)3 with either the N or Cterminus of the heavy chain of the Fab fragment (Fig. 6) [55]. Note that the heavy chain was chosen for the marker protein cloning to avoid the formation of nonfunctional dimers of the light chains. The constructed multipurpose pPICFab vector contained SacI/XhoI and PstI/BstEII pairs of sites for simple subcloning of genes of the heavy and light chains, a hexahistidine fragment on the Cterminus for conven ient purification of the desired protein using the metal chelate affinity chromatography, and a NotI site for cloning a marker protein (such as HRP, green fluorescent protein (EGFP), luciferase, etc.) on the Cterminus of the heavy chain of the antibody. To study the possibility of this approach, first the recombinant HRP fused with Fabfragments of the anti bodies against atrazine was obtained (Fig. 6). The yield of BIOCHEMISTRY (Moscow) Vol. 80 No. 4 2015

the fusion protein constituted approximately 3 to 10 mg per liter of culture liquid of P. pastoris. A relatively low yield of the secreted fusion protein in P. pastoris correlat ed with the yield for the separate expression of the HRP gene [55]. One of the factors negatively affecting the yield could be excessive glycosylation of the peroxidase part of the fusion protein, which is characteristic for P. pastoris cells. To test this hypothesis, the removal of the Nglyco sylation sites in the HRP gene could be helpful, or change in the HRP for another reporter protein, for example, green fluorescence protein. The antigenbinding activity of the recombinant fusion proteins was investigated by indirect competitive ELISA with atrazine. The results confirmed the presence of both peroxidase and antigenbinding activity in all the types of the fusion proteins. However, low activity of HRP–Fab compared to that of the Cterminal Fab–HRP fusion protein suggests that the spatial orientation of two components of the chimeric protein in the first case decreases the catalytic activity of the peroxidase. A typical calibration curve (Fig. 7) demonstrated a wide concentration range of atrazine detection (from 0.1 to 50 ng/ml), the variation coefficient not exceeding 8%. The IC50 value constituted 3 ng/ml, this being in agree


GRIGORENKO et al. was demonstrated. The recombinant conjugates exhibited both catalytic and antigenbinding activities. Thus, the use of contemporary methods of protein engineering for construction of horseradish peroxidase opens new opportunities for the development of a highly sensitive new generation of immuno and biosensors based on recombinant technology.

1.6 1.4


1.2 1.0 0.8

REFERENCES 0.6 0.4 0.2 0.01




[Atrazine], ng/ml

Fig. 7. Calibration curve for determination of atrazine by ELISA using Fab–HRP recombinant fusion protein.

ment with previous experimental data on determination of atrazine in the twostep ELISA procedure using Fab fragments of the same K411B antibodies [55], as well as with data on singlechain miniantibodies (scFv) expressed in E. coli [54, 56]. Thus, the recombinant fusion protein containing HRP and Fabfragments of the antibodies against atrazine [55] are functionally active and can be employed for the detection of atrazine by the ELISA procedure. Success in gene cloning allows construction of enzymes and fusion proteins with desirable properties by means of sitedirected and random mutagenesis. The introduction of a hexahistidine sequence (6× His) in the Cterminal region of HRP allowed efficient expression of the HRP gene in E. coli cells. A new procedure for refold ing and reactivation of the recombinant HRP from inclu sion bodies increased the yield of the active protein and significantly simplified the production of the recombi nant HRP. Gold electrodes modified with recombinant HRP exhibit high and stable amperometric signal of bioelectro catalytic reduction of hydrogen peroxide due to efficient direct electron transfer between the gold surface and the active site of the enzyme, which is the basis for the cre ation of a universal sensor for the detection of H2O2 as well as bienzyme sensors based on coimmobilization of rHRP and corresponding H2O2producing oxidases (lysine oxidase, glucose oxidase, etc.). The sensitivity of gold electrodes modified with recombinant HRP was 8 9fold higher than that for the native HRP. For the first time, the possibility of reproducible pro duction of recombinant functionally active conjugates of protein antigens and Fabfragments with HRP as the marker enzyme for application in enzyme immunoassays

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BIOCHEMISTRY (Moscow) Vol. 80 No. 4 2015

Recombinant horseradish peroxidase: production and analytical applications.

Horseradish peroxidase is a key enzyme in bio- and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli ...
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