Methods for Producing Heme Peroxidase

Abstract

The present invention provides a method for producing heme peroxidase from inclusion bodies (IBs), comprising the steps of: providing the heme peroxidase in the form of IBs; solubilizing the IBs; transferring the solubilized IBs into a refolding buffer to obtain a refolding mix; and adding a heme cofactor to the refolding mix, wherein the addition of the heme cofactor to the refolding mix is ​​distributed over a period of at least one hour. The present invention further provides a method for producing a heme peroxidase product.

Description

[Technical field]

[0001] The present invention relates to a method for producing heme peroxidase. [Background technology]

[0002] Heme peroxidases are an industrially important class of enzymes with a wide range of applications, including immunoassays, diagnostic kits, probe-based assay techniques such as ELISA, EMSA, Western blotting and Southern blotting, waste treatment, as reagents in organic synthesis, and potential therapeutic applications. A well-known member of this class of heme peroxidases is horseradish peroxidase (HRP), which is commonly used in research and industry.

[0003] So far, heme peroxidases have often been produced from plants, for example, HRP is mainly produced by extraction from hairy root cultures of horseradish (Armoracia rusticana). However, this is an inefficient and time-consuming process. Drawbacks include low yields, long culture times, seasonal availability, heterogeneous isoenzyme composition (different preparations have different biochemical properties), dependence on conditions during root growth, and plant glycosylation patterns that may be immunogenic in humans and consequently unsuitable for medical use.

[0004] Recombinant production of heme peroxidase is a desirable alternative since it is a more promising production method that guarantees a stable supply of defined enzyme preparations with high quality. In particular, recombinant production in E. coli may make it possible to obtain defined preparations of a single isoform with stable biochemical properties while lacking glycosylation and hence immunogenicity.

[0005] However, recombinant production of native heme peroxidases is challenging. For example, HRP contains glycosylation at eight asparagine residues, making recombinant production difficult. Wild-type HRP produced in E. coli exhibits poor stability compared to the plant-derived enzyme.

[0006] Numerous attempts have been made to recombinantly produce heme peroxidase in various hosts (mammalian cells, insect cells, yeasts (Pichia pastoris and Saccharomyces cerevisiae), other plants (Nicotiana tabacum, Nicotiana benthamiana, Armorasia lafatifolia) and Escherichia coli). However, these production strategies have resulted in very low yields and / or reduced enzyme activity and stability. In E. coli production, this occurs due to intracellular production of the protein, which causes inclusion body formation due to reducing conditions in the E. coli cytoplasm. This downstream process is therefore more complicated than in, for example, yeast. The enzymes mentioned above can also be transported to the periplasm by adding a signal sequence, but in this case the yields are even lower and the addition of a transport tag can impair the activity and stability of the enzyme. Also, E. coli cannot perform post-translational modifications, so the enzyme is not glycosylated, resulting in reduced protein stability.

[0007] Several studies have been conducted in the past with the aim of improving the properties of recombinantly produced HRP. An overview of attempts to improve HRP properties by mutation is provided by Humer and Spadiut ("Improving the performance of horseradish peroxidase by site-directed mutagenesis." International Journal of Molecular Sciences 20.4 (2019): 916), who disclose the effect of a number of mutations on stability and / or enzyme activity. Summary of the Invention

[0008] However, despite all these efforts, new and improved methods for producing heme peroxidase are still lacking. In particular, there is a need for methods for recombinantly producing and purifying heme peroxidase in a suitable host, in particular E. coli. It is an object of the present invention to provide such a method.

[0009] Accordingly, the present invention provides a method for producing heme peroxidase from inclusion bodies (IBs), comprising: - providing said heme peroxidase in the form of IB; - solubilizing the IBs; - transferring the solubilized IBs into a refolding buffer to obtain a refolding mix; - adding a heme cofactor to the refolding mix wherein addition of the heme cofactor to the refolding mix is ​​distributed over a period of at least 1 hour, preferably at least 2 hours, more preferably at least 3 hours, even more preferably at least 6 hours, in particular at least 10 hours.

[0010] Additionally, the present invention provides a method for producing a heme peroxidase product comprising producing a heme peroxidase according to the present invention.

[0011] In the context of the present invention, it has surprisingly been found that the refolding process of heme peroxidase can be strongly improved when the addition of the heme cofactor to the refolding mix is ​​distributed over a certain period of time (see Example 3, in particular comparison of Tables 5 and 6). Without being bound to a particular theory, the inventors speculate that this beneficial effect is due to the hydrophobic nature of the heme cofactor. Since this cofactor needs to form the holoenzyme, the refolding yield depends on the free heme cofactor being available for incorporation into the correctly folded heme peroxidase after refolding. Due to the hydrophobic nature of the cofactor, it can form aggregates that are not available for incorporation if added in one step.

[0012] As described in more detail herein below, further advantageous effects have been observed when the refolding mix is ​​incubated for a certain period of time before adding the heme cofactor (see in particular FIG. 1), when solubilization and refolding buffers with certain redox properties and pH values ​​are used (see Examples 2-5), when a precipitation step with certain salt concentrations is included after refolding (see Example 4, Table 9), and when the protein is further purified by hydrophobic interaction chromatography (HIC) (see Example 4). Importantly, all of these individual features may have advantageous effects independently of each other. Also, when some of these, or especially all of these, are combined, a highly optimized and efficient process is obtained (see in particular Example 5).

[0013] In addition to this new and improved production process, new HRP variants are disclosed herein that not only provide improved thermostability and kinetic parameters over wild-type HRP and known HRP variants, but are also particularly well suited for recombinant production in bacteria, particularly using the methods according to the invention, allowing the combination of a powerfully improved heme peroxidase and a highly efficient production process.

[0014] Techniques for protein expression and refolding from IBs are well established and commonly used in the art. Reviews of such techniques are provided, for example, by Cabrita and Bottomley ("Protein expression and refolding - A practical guide to getting the most out of inclusion bodies." Biotechnology Annual Review 10 (2004): 31-50) and Yamaguchi and Miyazaki ("Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. " Biomolecules 4.1 (2014): 235-251). Overexpression of recombinant proteins in bacteria such as E. coli often results in the formation of IBs, which are protein aggregates with non-native conformations. The term "inclusion bodies" (IBs) refers to insoluble aggregates containing heterologous polypeptides (e.g., the heme peroxidase) present in the cytoplasm of transformed host cells. These typically appear as bright spots under a microscope and can be recovered by cytoplasmic separation. Such inclusion bodies usually contain relatively pure and intact proteins; however, the inclusion bodies must be solubilized and the proteins must be refolded into their native structure.

[0015] Typically, IBs are solubilized in a solubilization buffer containing a denaturing agent such as urea, guanidinium chloride (GdnHCl), or an ionic detergent such as N-lauroyl sarcosine. Often, reducing agents such as 2-mercaptoethanol (β-ME), dithiothreitol (DTT) or 1-monothioglycerol (MTG) are added to reduce non-native intra- and intermolecular disulfide bonds and keep cysteines in a reduced state. The term "solubilization" or "solubilizing" refers to the process required to dissolve the IBs, resulting in monomolecular dispersion of the polypeptides with minimal intra- and intermolecular interactions. A "solubilization buffer" is a buffer suitable for solubilizing the IBs, i.e., typically a buffer in a state that is sufficiently denaturing for the IBs to be solubilized.

[0016] Refolding of denatured proteins (solubilized IBs) to active proteins (folded forms) typically occurs by removal of denaturants and is a key step in the efficient recovery of such proteins. The term "refolding" refers to the mechanism by which denatured proteins obtain their native or active conformation. Several approaches for refolding are known in the art. Refolding by dilution is one preferred method due to its simplicity, especially at production scale. Protein and denaturant concentrations are reduced in a single step, preventing aggregation due to intermolecular interactions. As used herein, "refolding buffer" refers to a buffer that is suitable for refolding purposes, i.e., a buffer that provides conditions that allow the denatured protein to obtain its native or active conformation. The refolding efficiency (yield) of the refolded protein can be estimated by biological activity, such as enzyme activity.

[0017] Many methods for producing and purifying heme peroxidases from inclusion bodies have been described in the art. A review of such techniques is provided by Eggenreich et al. (“Production strategies for active heme-containing peroxidases from E. coli inclusion bodies - a review.” Biotechnology Reports 10 (2016): 75-83). Such processes have been described for the production of a wide range of heme peroxidases, all of which may use the same basic process as outlined above (IB production, solubilization, refolding). As an example, horseradish peroxidase (Smith et al. “Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca 2+and heme.” Journal of Biological Chemistry 265.22 (1990): 13335-13343), fungal dye-decolorizing peroxidase (Linde et al. “Heterologous expression and physicochemical characterization of a fungal dye-decolorizing peroxidase from Auricularia auricula-judae.” Protein Expression and Purification 103 (2014): 28-37), cationic cell wall peroxidase (Kim et al. “Optimized refolding and characterization of S-peroxidase (CWPO_C of Populus alba) expressed in E. coli.” Protein Expression and Purification 80.2 (2011): 268-273), and Arabidopsis peroxidase (Shigeto et al. “Catalytic profile of Arabidopsis peroxidases, AtPrx-2, 25 and 71, contributing to stem lignification.” PLoS One 9.8 (2014)) and anionic tobacco peroxidase (Zakharova et al. “High-yield reactivation of anionic tobacco peroxidase overexpressed in Escherichia coli.” Protein Expression and Purification 113 (2015): 85-93).

[0018] WO2014 / 128726A2 describes a process for refolding recombinant proteins from IB; however, this is independent of peroxidase or the heme cofactor.

[0019] Lin et al. ("High yield production of fungal manganese peroxidases by E. coli through soluble expression, and examination of the activities." Protein expression and purification 145 (2018): 45-52) describe the expression of fungal manganese peroxidase in soluble form in E. coli. A method is described in which hemin is continuously added to the bacterial cell culture to increase hemin uptake into the cells, completely independent of refolding from IBs, and to avoid disturbance of proper cellular activity during protein expression.

[0020] Heme peroxidases are heme-containing enzymes capable of catalyzing oxidation reactions using hydrogen peroxide as an electron acceptor. In the context of the present invention, said heme peroxidases are preferably enzymes of EC 1.11.1.7. A major superfamily of heme peroxidases, also preferred in the context of the present invention, is the superfamily of plant, fungal and bacterial peroxidases (see Welinder "Superfamily of plant, fungal and bacterial peroxidases." Current Opinion in Structural Biology 2.3 (1992): 388-393). This superfamily is often viewed as consisting of three main classes: class I, the intracellular peroxidases; class II, consisting of secreted fungal peroxidases; and class III, consisting of secreted plant peroxidases. Classes II and III are classified as heme peroxidases, whose members typically have a heme group, four disulfide bridges, two Ca2+ ligands, and two Ca2+ ligands. 2+They are similar in binding ions. Also, members of both classes are usually glycosylated in the native state and are difficult to express in E. coli. As the advantageous effect of distributing the addition of the heme cofactor over a certain period of time in relation to the method of the invention is, inter alia, a result of the properties of this cofactor, the method of the invention may be advantageously used for the refolding of all heme peroxidases. However, class II and class III heme peroxidases are particularly preferred, as these classes are particularly difficult to recombinantly express. Thus, in a preferred embodiment, the heme peroxidase is a class II or class III heme peroxidase, in particular a class III heme peroxidase.

[0021] The heme cofactor used in the context of the present invention may be heme itself or any heme precursor or derivative that is incorporated into the apo-peroxidase. For example, hemin (the ferric chloride species of heme; CAS[16009-13-5]) is frequently used as a heme cofactor (see the literature related to methods for producing and purifying heme peroxidase from inclusion bodies listed herein above). In a preferred embodiment of the present invention, the heme cofactor is therefore hemin.

[0022] In the context of the present invention, it is particularly preferred when said heme peroxidase is horseradish peroxidase (HRP), which is a mutant HRP that has been optimized for recombinant expression in E. coli and / or has improved properties, such as improved thermostability and / or improved catalytic properties.

[0023] It has been surprisingly found that the properties of HRP can be improved by introducing mutations at proline-146 (P146) and / or asparagine-275 (N275) of wild-type HRP. In particular, it has been found that such HRP variants can be recombinantly produced in E. coli with high yields (especially using the method according to the invention) and good purity, and exhibit surprisingly good thermostability (see Example 7) and surprisingly high enzymatic activity (see Example 8). Although many mutations of HRP have been studied (see Humer and Spadiut, "Improving the performance of horseradish peroxidase by site-directed mutagenesis." International Journal of Molecular Sciences 20.4 (2019): 916), the above-mentioned substitutions of amino acids P146 and / or N275 have not been described in the prior art.

[0024] The sequence of wild-type HRP is known in the art, for example, from Gajhede et al. ("Crystal structure of horseradish peroxidase C at 2.15 Å resolution." Nature Structural Biology 4.12 (1997): 1032-1038, Protein Data Bank PDB ID 1ATJ) or UniProt entry P00433. Unless otherwise specified, residue numbering used herein refers to the sequence of wild-type HRP as set forth in SEQ ID NO:1 (positions P146 and N275 are shown below in bold).

[0025] (Wild type HRP (SEQ ID NO: 1):) [ka]

[0026] Plant-derived enzymes undergo post-translational modifications that result, among other things, in a free N-terminus with no added methionine. However, when recombinantly produced, for example in E. coli, they are typically produced with an N-terminal methionine residue derived from the start codon. Thus, recombinantly produced wild-type HRP typically has the following sequence:

[0027] (Recombinantly produced wild-type HRP (SEQ ID NO: 2): [ka]

[0028] In an embodiment of the invention, the heme peroxidase comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. Even more preferably, the heme peroxidase is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2. Particularly good results are obtained when the heme peroxidase is a polypeptide comprising an amino acid sequence which comprises at least two amino acid exchanges compared to SEQ ID NO: 1, said at least two amino acid exchanges being exchanges of amino acids P146 and N275 of SEQ ID NO: 1. As can be seen from the experimental results provided in Example 7, the combination of amino acid exchanges at both P146 and N275 (in HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K) can lead to unexpectedly higher thermostability than exchanges at single positions (HRP N13D / N57S / P146Q / N175S / N255D / N268D or HRP N13D / N57S / N175S / N255D / N268D / N275K).

[0029] In the context of the present invention, it is particularly preferred when said heme peroxidase comprises an amino acid sequence which comprises at least one amino acid exchange compared to SEQ ID NO: 1, said at least one amino acid exchange being selected from the group consisting of P146Q, P146A, P146R, P146V, P146E, N275K, N275R, N275D, N275S, N275Q, N275A and N275E. P146Q and N275K are particularly preferred.

[0030] It is further preferred when said amino acid sequence comprises an amino acid exchange selected from the group consisting of P146Q, P146A, P146R, P146V and P146E, in particular P146Q, compared to SEQ ID NO: 1. It is further preferred when said amino acid sequence comprises an amino acid exchange selected from the group consisting of N275K, N275R, N275D, N275S, N275Q, N275A and N275E, in particular N275K, compared to SEQ ID NO: 1. It is most preferred when one of said amino acid exchanges preferred for P146 (in particular P146Q) and one of said amino acid exchanges preferred for N275 (in particular N275K) are combined. As can be seen from the experimental results provided in Examples 7 and 8, such a combination can lead to both higher thermostability (in HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275 compared to HRP N13D / N57S / N175S / N255D / N268D / N275 or HRP N13D / N57S / N175S / N255D / N268D / N275K) and higher enzymatic activity (in HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275 compared to HRP N13D / N57S / N175S / N255D / N268D). In the context of the present invention, particularly good results have been observed when the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3. Said sequence comprises the amino acid exchanges P146Q and N275K, as well as the amino acid exchanges N13D, N57S, N175S, N255D and N268D (all marked in bold below) relative to SEQ ID NO:1.

[0031] (HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 3):) [ka]

[0032] As described above for wild-type HRP, when produced recombinantly, for example in E. coli, the mutant HRPs are typically produced with an N-terminal methionine residue derived from the start codon. Thus, the HRP mutants described above are typically produced recombinantly as proteins having the following sequences:

[0033] (Recombinantly produced HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4):)

[0034] [ka]

[0035] As mentioned above, typically the variants used in the context of the present invention contain an N-terminal methionine. However, in all cases, the amino acid numbering used by the present invention is applied according to the wild-type sequence (SEQ ID NO: 1) lacking an N-terminal methionine, i.e. this numbering applies correspondingly to the variants containing an N-terminal methionine. This means, for example, that in SEQ ID NO: 4, the mutation N13D is at (absolute) position 14, etc. This is also applicable to further variant embodiments with deletions or insertions.

[0036] In the context of the present invention, said heme peroxidase is preferably a polypeptide having peroxidase activity and comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 3, said amino acid sequence comprising at least one amino acid exchange compared to SEQ ID NO: 1, said at least one amino acid exchange being an exchange of amino acid P146 or amino acid N275 of SEQ ID NO: 1.

[0037] In a preferred embodiment, the heme peroxidase comprises an amino acid sequence having at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, particularly at least 98%, and most preferably at least 99% sequence identity with SEQ ID NO: 3. In order of increasing preference, the amino acid sequence has at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 3. Particularly preferred is when the amino acid sequence is the sequence set forth in SEQ ID NO: 3. Most preferred is when the heme peroxidase consists of the amino acid sequence set forth in SEQ ID NO: 4.

[0038] It is further preferred when the amino acid sequence further comprises at least one, preferably at least two, more preferably at least three, even more preferably at least four, especially five amino acid exchanges selected from the group consisting of N13D, N57S, N175S, N255D and N268D compared to SEQ ID NO:1.

[0039] The mutations N13D, N57S, N255D and N268D are disclosed by Capone et al. ("Glyco-variant library of the versatile enzyme horseradish peroxidase." Glycobiology 24.9 (2014): 852-863) and by Humer and Spadiut ("Improving the performance of horseradish peroxidase by site-directed mutagenesis." International Journal of Molecular Sciences 20.4 (2019): 916). Independently, but only for expression in yeast, the mutation N175S is disclosed by Morawski et al. ("Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae." Biotechnology and Bioengineering 76.2 (2001): 99-107).

[0040] All the above mutations N13D, N57S, P146Q, N175S, N255D, N268D and N275K with respect to SEQ ID NO: 1 are preferred in the context of the present invention, both individually and in combination with each other. It has been surprisingly found that the combination of the five mutations N13D, N57S, N175S, N255D and N268D leads to a particularly high thermostability (see Example 6). Also, the further combination of the hitherto completely unknown mutations P146Q and N275K leads to a significant improvement in enzyme activity (see Example 3; SEQ ID NO: 4).

[0041] Several further mutations of native HRP are known in the art and are also preferred in the context of the present invention. For example, Ryan et al. ("Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide." Biochimie 89.8 (2007): 1029-1032) describe the beneficial effects of the mutations K232N and T110V. In a preferred embodiment, the amino acid sequence thus further comprises the amino acid exchange T110V or K232N compared to SEQ ID NO: 1.

[0042] In the context of the present invention, "peroxidase activity" refers to the enzyme activity produced by the reaction of the substrates 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), or hydrogen peroxide (H 2 O 2 ) at pH 5 and 30° C. 2 O 2 When measured in a 50 mM phosphate-citrate buffer containing -1 s -1 , preferably at least 0.1 mM -1 s -1 , more preferably at least 1 mM -1 s -1 , and even more preferably at least 10 mM -1 s -1 , and even more preferably at least 20 mM -1 s -1 , and even more preferably at least 100 mM -1 s -1 , and even more preferably at least 1000 mM -1 s -1 , and even more preferably at least 10000 mM -1 s -1 , in particular at least 20000 mM -1 s -1In another preferred embodiment, the enzyme has a peroxidase activity corresponding to a kcat / Km value of 1 mM H 2 O 2 When measured in a 50 mM phosphate-citrate buffer containing -1 s -1 , preferably at least 1 mM -1 s -1 , more preferably at least 10 mM -1 s -1 , and even more preferably at least 100 mM -1 s -1 , most preferably at least 250 mM -1 s -1 In yet another preferred embodiment, the heme peroxidase has a catabolic activity of 10 mM ABTS at pH 5 and 30° C. when measured in 50 mM phosphate-citrate buffer. 2 O 2 The peroxidase activity corresponding to a kcat / Km value of at least 1 mM -1 s -1 , preferably at least 10 mM -1 s -1 , more preferably at least 100 mM -1 s -1 , and even more preferably at least 1000 mM -1 s -1 , most preferably at least 2500 mM -1 s -1 Preferably, the peroxidase activity is measured as described in Example 6. A 50 mM phosphate-citrate buffer can be obtained by adding 25.7 ml of 0.2 M dibasic sodium phosphate and 24.3 ml of 0.1 M citric acid to 50 mL of deionized water.

[0043] It is further preferred that the peroxidase activity of said heme peroxidase is at least 10%, preferably at least 20%, more preferably at least 50% of the peroxidase activity of wild-type HRP, in particular of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 2, especially when produced and measured under the same conditions.

[0044] It is further preferred when said heme peroxidase has increased thermostability relative to wild-type HRP. It is preferred when, for said amino acid sequence having a certain percent sequence identity with SEQ ID NO:3 as defined above, a polypeptide consisting of such amino acid sequence has increased thermostability relative to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, preferably SEQ ID NO:2. A methionine residue may be added to the N-terminus of said amino acid sequence having a certain percent sequence identity with SEQ ID NO:3 so that it may be recombinantly produced in E. coli. That is, it is preferred when a polypeptide consisting of the above-mentioned amino acid sequence followed by an N-terminal methionine has increased thermostability relative to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2. To compare the thermostabilities of such two polypeptides, said polypeptides may be produced and measured under the same conditions. "Increased thermostability" preferably means a longer half-life when incubated at 60°C in a buffer consisting of 20 mM BisTris / HCl (pH 7), 7% glycerol and 500 mM NaCl. Preferably, the half-life of the polypeptide consisting of the above mentioned amino acid sequence (and / or of said heme peroxidase comprising said amino acid sequence) is at least 1.5 times, preferably at least 3 times, more preferably at least 6 times, even more preferably at least 12 times higher than the polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 2. Preferably, said half-life is at least 1.5 times, preferably at least 3 times, more preferably at least 6 times, even more preferably at least 12 times higher than the polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 1 or ... 2 O 2The half-life is determined by measuring the residual peroxidase activity using 7 mM ABTS in 50 mM phosphate-citrate buffer containing 10 mM phosphate-citrate. Preferably, for half-life measurements, the concentration of the polypeptide is 2.86 μM. Preferably, the half-life is determined as described in Example 6.

[0045] In a preferred embodiment, the heme peroxidase and / or polypeptide consisting of the amino acid sequence having a certain percent sequence identity with SEQ ID NO: 3 as defined above is dissolved in 1 mM H at pH 5 and 30° C. 2 O 2 The half-life at 60° C. in a buffer consisting of 20 mM BisTris / HCl (pH 7), 7% glycerol and 500 mM NaCl, as measured by residual peroxidase activity using 7 mM ABTS in 50 mM phosphate-citrate buffer containing is at least 0.5 hours, preferably at least 1 hour, more preferably at least 2 hours, even more preferably at least 4 hours, and most preferably at least 6 hours.

[0046] With regard to the method of the invention for producing heme peroxidase, it has surprisingly been found that distributing the addition of the heme cofactor to the refolding mix over a certain period of time leads to superior results compared to adding all the cofactor at once in one batch (see Example 3; comparison of Tables 5 and 6). Generally, the slower the addition of the heme cofactor, the better. The addition of the heme cofactor to the refolding mix is ​​preferably distributed over a period of at least 1 hour, with longer periods being even more preferred. In order of increasing preference, the period is at least 2, 3, 4, 5, 6, 7, 8, 9, and in particular at least 10 hours. In this context, "distributed over a period of at least 1 hour" can mean that a constant feed of the heme cofactor is applied over a period of at least 1 hour, or that the total amount of heme cofactor is divided into several smaller doses, for example one dose every 10 minutes or every hour, over a period of several hours. The same applies correspondingly to other periods (e.g. in a preferred embodiment where the addition is distributed over a period of at least 10 hours, a portion of the cofactor can be added once per hour over a period of at least 10 hours, or as a constant feed over at least 10 hours). The addition of the heme cofactor preferably comprises at least two (but preferably at least three, more preferably at least four, even more preferably at least six, most preferably at least 10) increases in the heme cofactor concentration, the first and last increases being at least separated by the periods specified above (e.g. at least two increases in the heme cofactor concentration separated by at least one hour, preferably at least four hours, in particular at least 10 hours). This can be achieved either by multiple separate heme cofactor concentration increase steps (wherein the total amount of heme cofactor added is divided into multiple smaller doses) or by a continuous supply of heme cofactor, or by a combination of the two. It is particularly advantageous when the total amount of heme cofactor added to the refolding mix is ​​evenly distributed.Thus, it is preferred when no more than 80% of the total amount of heme cofactor added to the refolding mix is ​​added in a period of less than 1 hour, preferably no more than 70%, more preferably no more than 60%, even more preferably no more than 50%, even more preferably no more than 40%, even more preferably no more than 30%, even more preferably no more than 20%, and most preferably no more than 10%.

[0047] It is particularly preferred when the heme cofactor is added to the refolding mix as a continuous feed, as this allows for a particularly slow and well-distributed addition of the heme cofactor. It is particularly advantageous when the concentration of the heme cofactor provided as the heme cofactor feed is constant over time.

[0048] In a preferred embodiment of the method of the invention, the heme cofactor is added to the refolding mix at a rate of 0.1-10 μmol / L / h, preferably 0.2-5 μmol / L / h, even more preferably 0.5-3 μmol / L / h, even more preferably 1-2.5 μmol / L / h, in particular 1.4-1.8 μmol / L / h. It is preferred that the heme cofactor is added at a rate of 0.01-1 molar equivalent / h, preferably 0.02-0.5 molar equivalent / h, more preferably 0.05-0.2 molar equivalent / h, relative to the heme peroxidase present in the refolding mix. The rate is preferably an average rate, i.e. when the heme cofactor is applied in separate steps over a certain period of time, the average rate can be calculated by dividing the total amount of heme cofactor by the period between the first and last step.

[0049] In the context of the present invention, it has been found that results can be even further improved when the heme cofactor is not added immediately to the refolding mix, in particular when it is not already present in the refolding buffer. Instead, it has been found to be advantageous when the heme cofactor is added only after incubating the refolding mix for a certain period of time (see in particular Example 2, DoE4 and FIG. 1). It has therefore been found to be advantageous when the refolding can proceed for at least a certain period of time in the absence of the heme cofactor. In a preferred embodiment of the present invention, the refolding mix is ​​therefore incubated for at least 1 hour, preferably at least 2 hours, even more preferably at least 4 hours, in particular at least 8 hours, before the addition of the heme cofactor. This does not, however, mean that there can be no heme cofactor at all during this incubation time; for this embodiment, it is simply required that the heme cofactor is added to the refolding mix after the specified period of time. Preferably, however, during said period, said heme cofactor is present in the refolding mix at less than 10 μM, preferably less than 2 μM, even more preferably less than 0.5 μM, most preferably less than 0.1 μM.

[0050] In a preferred embodiment, at least 1 μmol / L, preferably at least 4 μmol / L, even more preferably at least 10 μmol / L, in particular at least 20 μmol / L of the heme cofactor is added to the refolding mix. Preferably, 1 μmol / L to 200 μmol / L, more preferably 4 μmol / L to 100 μmol / L, even more preferably 10 μmol / L to 50 μmol / L, in particular 15 μmol / L to 25 μmol / L of the heme cofactor is added to the refolding mix. It is particularly preferred when at least 0.25 molar equivalents, preferably at least 0.5 molar equivalents, even more preferably at least 1 molar equivalent, in particular at least 2 molar equivalents of the heme cofactor are added to the refolding mix relative to the heme peroxidase. In particular, 0.25 to 10 molar equivalents, preferably 0.5 to 5 molar equivalents, even more preferably 1 to 4 molar equivalents, especially 1 to 2 molar equivalents of the heme cofactor relative to the heme peroxidase may be added to the refolding mix.

[0051] Standard techniques known in the art can be used to obtain the IBs used in the method according to the invention. The skilled artisan is familiar with how to recombinantly express a particular heme peroxidase in a suitable host cell, e.g. E. coli, to obtain IBs containing said heme peroxidase. Preferably, said heme peroxidase in the form of IBs is provided by: culturing a host cell expressing a gene encoding said heme peroxidase; and obtaining IBs from said host cell. Preferably, said host cell is a prokaryotic cell, preferably an E. coli cell.

[0052] Due to its advantageous properties, the method according to the invention is well suited for the production of heme peroxidase in large quantities. As demonstrated in the examples described herein, the method of the invention is scalable to larger culture volumes and therefore to higher amounts of heme peroxidase production. In a preferred embodiment, the cells are therefore cultured in a volume of at least 2L, preferably at least 5L, more preferably at least 20L, even more preferably at least 50L, in particular at least 100L.

[0053] In the course of the invention, it has turned out that the method for producing heme peroxidase can be improved even further by using a specific composition of solubilization and refolding buffers (see Examples 2-5). Preferably, solubilizing the IBs comprises incubating the IBs in a solubilization buffer. As described in the Examples, it has been found that a certain pH of the solubilization buffer leads to particularly advantageous results (see in particular Example 2, Table 3, and Figures 2 and 3). In particular, it is preferred when the solubilization buffer has a pH of at least 8, preferably at least 8.5, more preferably at least 9.0, even more preferably at least 9.5. It is further preferred when the solubilization buffer has a pH of 8 to 12.5, preferably 8.5 to 11.5, more preferably 9 to 11, even more preferably 9.5 to 10.5.

[0054] Preferably, the solubilization buffer used to solubilize the IBs contains a reducing agent. In the context of the present invention, it has been found that certain reducing agent concentrations lead to particularly advantageous results (see in particular Example 2, DoEs 1-3; Example 3, Reactor Runs 1-4). Preferably, the reducing agent is DTT. However, other reducing agents can be used as well. The skilled person is familiar with other reducing agents commonly used for IB solubilization, such as β-ME, TCEP (tris(2-carboxyethyl)phosphine), cysteine, etc. All of these reducing agents are also preferred in the context of the method of the present invention. It is preferred that the redox state in the solubilization buffer corresponds to a DTT concentration of 1-50 mmol / L, preferably 2-25 mmol / L, more preferably 4-15 mmol / L, in particular 6-8 mmol / L. In this context, the term "redox state" refers to the reduction potential, and "corresponding to" means that either DTT is present in the amount indicated, or another reducing agent (such as β-ME, TCEP or cysteine) is present in a concentration that results in the same reduction potential when measured in a solution of 50 mM glycine, 6 M urea in water (pH 10) at 25°C. Preferably, the reduction potential of the solubilization buffer is between -150mV and -500mV, preferably between -200mV and -450mV, more preferably between -250mV and -400mV, even more preferably between -280mV and -370mV, even more preferably between -300mV and -340mV, and most preferably between -310mV and -330mV. The reduction potential is preferably measured at a temperature of 25°C and a pressure of 101.300 Pa. The skilled person is familiar with how to measure the reduction potential. Preferably, the measurement of the reduction potential is carried out using an EasyFerm Plus ORP Arc425 connected to a Lucullus process system, preferably as described in Example 3.Alternatively, the measurement can be performed as described in standard ASTM D1498-14 ("Standard Test Method for Oxidation-Reduction Potential of Water", ASTM International, West Conshohocken, PA, 2014, www.astm.org).

[0055] In another preferred embodiment, the reducing agent contains a thiol group. Preferably, the concentration of thiol groups in the solubilization buffer is 2-100 mmol / L, preferably 4-50 mmol / L, more preferably 8-30 mmol / L, especially 12-16 mmol / L. The concentration of thiol groups in the solubilization buffer is preferably determined based on the concentration of the thiol-containing substance added to the solubilization buffer (e.g., DTT contains 2 thiol groups per molecule; a concentration of 1 mmol / L of DTT therefore corresponds to a concentration of 2 mmol / L of thiol groups). Alternatively, the concentration of thiol groups may also be determined experimentally. Preferably, the concentration of thiol groups in the solubilization buffer is measured using Ellman's reagent. Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid), or DTNB) is a chemical used to quantify the number or concentration of thiol groups in a sample. The skilled artisan is familiar with how to carry out the measurement using Ellman's Reagent. Preferably, the measurement is carried out as described by Simpson ("Estimation of Free Thiols and Disulfide Bonds Using Ellman's Reagent." CSH Protocols 2008 (2008): pdb-prot4699).

[0056] In addition to the reducing agent, the solubilization buffer typically contains a denaturing agent. The skilled person is familiar with suitable denaturing agents, for example urea or GdnHCl. Preferably, the solubilization buffer contains urea, preferably at a concentration of at least 4 mol / L, more preferably at least 5 mol / L, even more preferably at least 6 mol / L. Preferably, the solubilization buffer contains urea at a concentration of 4 mol / L to 8 mol / L, preferably 5 mol / L to 7 mol / L, in particular 5.5 to 6.5 mol / L.

[0057] It is preferred when the IBs are incubated in the solubilization buffer for at least 5 minutes, preferably at least 10 minutes, more preferably at least 15 minutes, especially at least 30 minutes to solubilize the IBs. Preferably, the IBs are incubated in the solubilization buffer at a temperature between 4°C and 40°C, preferably between 10°C and 35°C, even more preferably between 20°C and 25°C.

[0058] Advantageous results may be obtained when the solubilized IBs are mixed with the refolding buffer in a ratio of 1:5 v / v to 1:250 (solubilized IBs:refolding buffer), preferably 1:10 to 1:100, even more preferably 1:20 to 1:80, in particular 1:30 to 1:60. As mentioned above, the method of the invention is particularly well suited for producing the heme peroxidase in large quantities. The refolding is therefore preferably carried out in a volume of at least 5 L, preferably at least 10 L, more preferably at least 25 L, even more preferably at least 100 L. This means that the refolding mix preferably has a volume of at least 5 L, preferably at least 10 L, more preferably at least 25 L, even more preferably at least 100 L.

[0059] As in the case of the solubilization buffer, advantageous effects have been found for certain pH ranges of the refolding buffer (see in particular Tables 3, 7 and 14 and Figures 2 and 3). It is therefore preferred when the refolding buffer has a pH of at least 8, preferably at least 8.5, more preferably at least 9, even more preferably at least 9.5. In particular it is preferred when the refolding buffer has a pH of 8 to 12.5, preferably 8.5 to 11.5, more preferably 9 to 11, even more preferably 9.5 to 10.5. It is particularly preferred when the pH difference between the solubilization buffer and the refolding buffer is less than 2 pH increments, preferably less than 1 pH increments, more preferably less than 0.5 pH increments, even more preferably less than 0.2 pH increments, in particular less than 0.1 pH increments. This means, for example, that when the solubilization buffer has a pH of 10, the refolding buffer has a pH of preferably 9 to 11 (i.e., the difference is a pH increment of less than 1), more preferably 9.5 to 10.5 (the difference is a pH increment of less than 0.5), etc.

[0060] Preferably, the refolding buffer used in accordance with the method of the invention contains an oxidizing agent. The skilled person is familiar with oxidizing agents commonly used in refolding buffers, such as glutathione disulfide (GSSG) or cystine, all of which are preferred in accordance with the method of the invention. As demonstrated herein in the Examples (see, for example, Tables 1, 3 and 7, and Figures 2 and 3), advantageous effects have been observed with certain oxidizing agent concentrations. Preferably, the refolding buffer has a redox state corresponding to a GSSG concentration of 0.2-6 mM GSSG, preferably 0.4-4 mM GSSG, more preferably 0.8-2.5 mM GSSG, in particular 1.1-1.6 mM GSSG. In this context, the term "redox state" refers to the reduction potential, and "corresponding to" means that GSSG is present in the indicated amount, or that another oxidizing agent (such as cystine) is present in the indicated amount, such as 20 mM glycine, 2 mM CaCl 2or at a concentration that results in the same reduction potential when measured at 25° C. in a solution of 2 M urea, 7% v / v glycerol, pH 10. Preferably, the refolding buffer has a reduction potential of 50 mV to 110 mV, preferably 60 mV to 100 mV, more preferably 65 mV to 95 mV, most preferably 70 mV to 90 mV. Preferably, the reduction potential is measured as described above for the solubilization buffer.

[0061] It is further preferred that the refolding buffer contains a denaturant. The same denaturant as for the solubilization buffer is preferred, although at a lower concentration. Preferably, the refolding buffer contains 0.5-3 mol / L, preferably 1 mol / L-2.7 mol / L, even more preferably 1.5 mol / L-2.4 mol / L, in particular 1.8-2.2 mol / L of urea.

[0062] It is particularly preferred when the refolding mix contains both DTT and GSSG. Preferably, the molar ratio between DTT and GSSG in the refolding mix is ​​1:2 (DTT:GSSG) to 1:30, preferably 1:3 to 1:20, even more preferably 1:4 to 1:15, in particular 1:6 to 1:8. It should be understood that these ratios are calculated based on the initial amounts of DTT and GSSG added to the solubilization buffer and the refolding buffer, and that consumption of DTT and / or GSSG should not be taken into account.

[0063] Preferably, the refolding mix has a reduction potential of -105 mV to 135 mV, preferably -90 mV to 105 mV, even more preferably -80 mV to 80 mV, even more preferably -70 mV to 60 mV, and most preferably -60 mV to 50 mV. Preferably, the reduction potential is measured as described above for the solubilization buffer. Preferably, the reduction potential is measured immediately upon mixing the solubilization buffer and the refolding buffer, prior to addition of the heme cofactor.

[0064] In the context of the present invention, it has been found to be advantageous when a precipitation step involving high salt concentrations is included after refolding (see Example 4, Table 9). Surprisingly, it has been found that this method allows for effective removal of impurities without significant amounts of the heme peroxidase being precipitated. The method of the present invention therefore preferably further comprises a step of adding salt to the refolding mix. In a preferred embodiment, the salt is from 2 mol / L to 12 mol / L, preferably from 4 mol / L to 10 mol / L, even more preferably from 4.5 mol / L to 8 mol / L. Particularly good results have been obtained when the salt is sodium chloride, preferably at a concentration of 0.5 to 6 mol / L, more preferably from 1.5 to 5.5 mol / L, even more preferably from 2.5 to 5 mol / L, in particular from 3.5 to 4.5 mol / L. In another preferred embodiment in which good results have been obtained, the salt is ammonium sulfate, preferably at a concentration of 0.25-1.5 mol / L, preferably 0.5-1.4 mol / L, even more preferably 0.8-1.2 mol / L. Any salt commonly used to salt out proteins may be used in the context of the present method. The skilled person will be familiar with such salts, e.g., KCl, K 2 CO 3 , CaCl 2 , N.H. 4 Cl, Na 2 SO 4 , or NaOAc. The optimal concentration of such alternative salts can be based on the ionic strength of the resulting solution (I = 0.5 * Σ(c i * z i 2 ), where c i is the molar concentration of each ion, and z i is the charge number of the ion; the sum is taken over all ions contained in the salt; for example, 1 mol / L (NH 4 ) 2 SO 4 Then, the ionic strength is I=0.5 * (2mol / L * 1 2 +1mol / L* 2 2 )=3 mol / L). Preferably, the salt is added to the refolding mix to an ionic strength of 0.5-6 mol / L, preferably 1.5-5 mol / L, even more preferably 2.5-4.5 mol / L, especially 3.5-4.5 mol / L.

[0065] The salting-out strength of a particular salt can also be determined by its position in the so-called Hofmeister series (also called the "lyotropic series"). The Hofmeister series is a classification of ions in order of their ability to salt out or salt proteins (exemplary anions in order of decreasing salting-out strength are SO, 4 2- >HPO 4 2- >OAc - >Cl - >NO 3 - >I - >SCN - and similarly for the cation: NH 4 + >K + >Na + >Li + >Mg 2+ >Ca 2+>guanidinium). In the context of the present invention, it is preferred when the salt has a salting out strength between ammonium sulfate and sodium chloride as determined by its position in the Hofmeister series, preferably the concentration of the salt is 1.25-6 mol / L, more preferably 0.5-5 mol / L, even more preferably 1.0-4.5 mol / L, most preferably 1.5-4 mol / L. In a further preferred embodiment, the salt has a salting out strength weaker than sodium chloride as determined by its position in the Hofmeister series, preferably the concentration of the salt is at least 2 mol / L, preferably at least 4 mol / L, more preferably at least 4.5 mol / L. As used herein, a first salt is considered to have a higher / lower salting out concentration than a second salt when both the cation and anion of a first salt have a higher / lower salting out concentration than the cation and anion of a second salt as determined by the Hofmeister series.

[0066] Preferably, the method of the invention further comprises a centrifugation step to remove impurities from the refolded heme peroxidase. Alternatively or additionally, the method of the invention may further comprise a filtration step to remove impurities from the refolded heme peroxidase. These embodiments are particularly advantageous when salt is added to the refolding mix such that impurities are precipitated but (the majority of) the heme peroxidase remains in solution.

[0067] Preferably, the method according to the invention further comprises a step of purifying the refolded heme peroxidase. It has been found to be particularly advantageous when said heme peroxidase is in holo form during purification, and preferably said heme peroxidase is therefore purified after the heme cofactor has been added to the refolding mix.

[0068] For purification purposes, purification tags such as polyhistidine tags (typically consisting of several, e.g., 6-10, histidine residues, often added to the protein N- or C-terminus) are frequently used in the art. Such tags may also be used in the context of the present invention. However, the heme peroxidase is preferred when it does not comprise a purification tag. In particular, the heme peroxidase is preferred when it does not comprise a polyhistidine tag.

[0069] For the method of the invention, it is preferred that said heme peroxidase is purified by chromatography, in particular by hydrophobic interaction chromatography (HIC). As demonstrated by the examples, this type of purification leads to surprisingly good results, in particular high final concentrations, excellent purity and high activity (see Example 4). Since HIC typically involves binding conditions with high salt concentrations, HIC is particularly well suited for combination with a salting-out step to remove impurities, as described herein above. Thus, although such high salt concentrations may precipitate impurities and excess heme cofactor, correctly folded heme peroxidase remains in solution and can be used directly as load for HIC. The skilled person is familiar with how to perform HIC. It is preferably performed as described in Example 4. It is particularly preferred when the stationary phase is a butyl sepharose resin.

[0070] As used herein, a "heme peroxidase product" can be any product obtained from the heme peroxidase produced by the method according to the invention, for example, a conjugate of said heme peroxidase with another molecule (e.g., a protein), or an otherwise modified or derivatized heme peroxidase (e.g., a heme peroxidase linked to a solid support). A "heme peroxidase product" can also be any type of commercially available product containing a heme peroxidase (or a modified / derivatized / conjugated heme peroxidase), for example, a composition containing such a heme peroxidase (e.g., in solution or in lyophilized form), or a container containing such a composition, or a kit containing such a container, or any type of packaged product.

[0071] In many applications, heme peroxidases such as HRP are used as conjugates with other molecules. Such conjugates are useful in techniques such as, for example, Western blots, ELISAs, and immunohistochemistry. One type of widely used conjugate is a conjugate with an antibody or another binding protein. In this case, a substrate for HRP can be added to generate a detectable signal at the site where the binding protein binds to its target. Other frequently used HRP conjugates include HRP-streptavidin conjugates (e.g., for use in sandwich ELISA applications to detect biotinylated antibodies), or HRP-protein A, G, or L conjugates (proteins A, G, and L bind to immunoglobulins and are therefore useful for detecting primary antibodies in, for example, ELISA, ELISPOT, IHC, or Western blotting).

[0072] Many commercial suppliers offer kits for conjugating heme peroxidases such as HRP to proteins of interest (e.g., HRP Conjugation Kit / HRP Labeling Kit ab102890 from Abcam, LYNX Rapid HRP Antibody Conjugation Kit (product code LNK001P) from Bio-Rad, or EZ-Link Plus Activated Peroxidase Kit (catalog number 31489) from Thermo Fisher Scientific). The purpose of such kits is typically to allow the user to conjugate the peroxidase to any protein of interest, such as an antibody. Such kits may contain the heme peroxidase in an activated form (for direct reaction with another protein) or the kit may contain the heme peroxidase and a suitable cross-linker for conjugation by the user.

[0073] Therefore, in a preferred embodiment, the method of the invention further comprises a step of activating the heme peroxidase for a conjugation reaction. Preferably, the heme peroxidase is reacted with a cross-linking agent suitable for conjugating the heme peroxidase to another molecule, preferably another protein. Suitable techniques for conjugation and suitable cross-linking agents are known to the skilled artisan and can be found, for example, in the book Hermanson, Greg T. Bioconjugate Techniques. Academic press, 2013. Preferably, the cross-linking agent forms a covalent bond with the heme peroxidase. Preferably, the cross-linking agent comprises one, in particular two reactive groups selected from the group consisting of NHS ester, succinimidyl ester, imidoester, difluoro, haloacetyl, maleimide, pyridyldithiol and hydrazide. For example, the cross-linking agent is a bifunctional PEG linker having a maleimide group and an active ester group, such as maleimide-PEG. 8- succinimidyl ester (CAS number 756525-93-6, commercially available, for example, from Sigma-Aldrich, catalog number 746207).

[0074] Preferably, when the heme peroxidase is "activated for conjugation", this means that the heme peroxidase comprises a reactive group capable of forming a covalent bond with another protein, preferably with a binding protein, in particular with an antibody. It is particularly preferred that the heme peroxidase is covalently linked to a chemical crosslinker as described above, in particular that at least one reactive group of the crosslinker is available for reaction with another molecule, preferably with another protein. This can be achieved, for example, by reacting a further reactive group of the crosslinker with the heme peroxidase to form the covalent bond.

[0075] The heme peroxidase product may consist of the activated heme peroxidase for conjugation, packaged in a suitable container, e.g. a vial, in lyophilized form or in solution. However, the heme peroxidase and the crosslinker may also be provided separately. The heme peroxidase product may therefore be a kit comprising the heme peroxidase and the crosslinker. The method for producing the heme peroxidase product may therefore further comprise packaging the heme peroxidase and the above mentioned crosslinker in a kit. Preferably, the crosslinker is suitable for conjugating a polypeptide to another protein, preferably a binding protein.

[0076] The heme peroxidase produced by the method of the invention may be a polypeptide consisting of the amino acid sequence set out in SEQ ID NO: 4. However, said heme peroxidase may also be a polypeptide having peroxidase activity as defined herein above and further comprising the amino acid sequence of an additional protein. A fusion protein may be obtained by known methods, for example by creating a gene fusion (connecting a nucleic acid sequence encoding said heme peroxidase to a nucleic acid sequence encoding said additional protein, preferably by including a linker sequence between such two proteins). Said additional protein may be streptavidin. In another preferred embodiment, said additional protein is an antibody binding protein, preferably protein A, protein G or protein L. In a particularly preferred embodiment, the heme peroxidase produced by the method of the invention comprises the amino acid sequence of a binding protein. Said binding protein may be any protein that can act as a substance for binding to a molecule of interest (e.g. for detecting a specific protein or another molecule). Preferably, said binding protein is an antibody. In a further preferred embodiment, said binding protein is an antibody fragment, preferably a single chain variable fragment (scFv) or an antigen-binding fragment (Fab). In a further preferred embodiment, said binding protein is an antibody mimetic, preferably selected from the group consisting of adnectins, affibodies, anticalins, DARPins, engineered Kunitz-type inhibitors, and monobodies. Many such suitable binding proteins are known from the art, for example from Gebauer and Skerra ("Engineered protein scaffolds as next-generation antibody therapeutics." Current opinion in chemical biology 13.3 (2009): 245-255).

[0077] In a preferred embodiment of the method for producing a heme peroxidase product of the invention, the method further comprises a conjugation reaction to obtain a heme peroxidase conjugate. Preferably, the heme peroxidase is conjugated to a binding agent, preferably an antibody. In a further preferred embodiment, the binding agent is a binding protein, preferably any of the binding proteins listed above. It is particularly preferred when the heme peroxidase is conjugated to an antibody fragment, preferably a single chain variable fragment (scFv) or an antigen-binding fragment (Fab). It is further preferred when the heme peroxidase is conjugated to an antibody mimic, preferably selected from the group consisting of adnectins, affibodies, anticalins, DARPins, engineered Kunitz inhibitors, and monobodies. In another preferred embodiment, the heme peroxidase is conjugated to an antibody binding protein, preferably protein A, protein G, or protein L. In yet another preferred embodiment, the heme peroxidase is conjugated to a further protein, preferably streptavidin.

[0078] Conjugates between heme peroxidase and other proteins can be obtained by conjugation reactions known to those skilled in the art, for example by chemical crosslinking. For this purpose, the heme peroxidase and the other protein can be linked together by a chemical crosslinker that forms a stable, preferably covalent, link between the two molecules. Such methods are often called bioconjugation and are known in the art. Many suitable methods can be found, for example, in the book Hermanson, Greg T. Bioconjugate Techniques. Academic press, 2013.

[0079] The method of the invention for producing the heme peroxidase product may further comprise purification, concentration or other processing of the heme peroxidase. Preferably, the method further comprises the step of purifying the heme peroxidase or the heme peroxidase conjugate. The heme peroxidase may already be derivatized or otherwise modified in this regard. In a preferred embodiment, the method further comprises the step of freezing the heme peroxidase or the heme peroxidase conjugate. Alternatively or in combination with the above embodiment, the method preferably further comprises the step of lyophilizing the heme peroxidase or the heme peroxidase conjugate.

[0080] The heme peroxidase product may also be a composition comprising the heme peroxidase produced by the method according to the invention, or a product (e.g., a container) comprising said composition. Preferably, one or more additives are added to the composition. Such additives may, for example, further increase the stability of the heme peroxidase during storage or ensure a longer shelf life and a stable biological activity level. Preferred additives include pH buffers, stabilizers, bulking agents, tonicity adjusters, etc. Additives that are suitable for use in conjunction with lyophilization are particularly preferred.

[0081] Suitable additives are known to those skilled in the art. For example, the composition preferably contains a pH buffer, more preferably selected from the group consisting of glycine, histidine, glutamate, succinate, phosphate, acetate, and aspartate. It is further preferred that the composition contains a bulking agent, more preferably selected from the group consisting of mannitol, glycine, sucrose, dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol. The composition preferably comprises a stabilizer selected from the group consisting of sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL, and polyhydroxy compounds including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose and hyaluronic acid, and sodium chloride. The composition may include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, sodium dioctyl sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, glycodeoxycholic acid sodium salt, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-1 0, SB3-12, digitonin, TritonX-100, TritonX-114, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, soybean lecithin, DOPC, DMPG, DMPC, and DOPG; and may further comprise a surfactant preferably selected from the group consisting of sucrose fatty acid esters, methylcellulose and carboxymethylcellulose.

[0082] Advantageously, said composition comprises at least 0.01 mg, preferably at least 0.1 mg, more preferably at least 1 mg, even more preferably at least 5 mg, in particular at least 10 mg of the heme peroxidase produced according to the invention. It is further preferred when said composition contains said heme peroxidase in a concentration of at least 0.0001% (weight / weight, w / w), preferably at least 0.001% w / w, more preferably at least 0.01% w / w, even more preferably at least 0.1% w / w, even more preferably at least 1% w / w, in particular at least 10% w / w.

[0083] Advantageously, said composition is a solid composition (at 25° C. and at atmospheric pressure), preferably a lyophilized composition. In another preferred embodiment, said composition is a liquid composition (at 25° C. and at atmospheric pressure). Advantageously, said composition contains the heme peroxidase produced according to the invention in a concentration of at least 0.001 mg / mL, preferably at least 0.01 mg / mL, more preferably at least 0.1 mg / mL, even more preferably at least 0.5 mg / mL and most preferably at least 2 mg / mL.

[0084] It is further preferred that the composition is substantially free of DNA, in particular dsDNA. Preferably, the composition contains less than 1 μg / g DNA, preferably less than 100 ng / g, more preferably less than 10 ng / g, most preferably less than 1 ng / g. The DNA concentration can be determined by flowmetry using the dye SYBR Green I (N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine). The skilled artisan is familiar with measuring DNA concentration using flowmetry. The measurement can be performed as described in Rengarajan, Kalpana, et al. ("Technical Brief Quantifying DNA concentrations using fluorometry: A comparison of fluorophores." Molecular Vision 8 (2002): 416-421).

[0085] Said heme peroxidase product can find use in various therapeutic applications, for example in targeted cancer therapy. In a preferred embodiment, said heme peroxidase product is therefore a pharmaceutical composition, preferably comprising one or more additives, which are pharma- ceutically acceptable for administration to an individual, in particular a mammal, in particular a human. Suitable additives are known to those skilled in the art, for example water (in particular water for injection), saline, Ringer's solution, dextrose solution, buffers, Hank's solution, vesicle-forming compounds (e.g. lipids), fixed oils, ethyl oleate, 5% dextrose in saline, substances improving isotonicity and chemical stability, buffers, and preservatives. Other suitable additives include any compound that, when administered to a patient, does not induce the production of antibodies that are harmful to said patient. Examples are well-tolerated proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. This pharmaceutical composition is preferably suitable for parenteral administration, in particular for intravenous administration. The pharmaceutical composition may be provided as an injectable unit dosage form, e.g., a solution, suspension or emulsion, formulated together with a pharma- ceutically acceptable excipient as defined above. Generally, all the preferred embodiments listed above for the composition (especially those relating to the concentration and amount of the heme peroxidase) are also preferred when the heme peroxidase product is a pharmaceutical composition.

[0086] For therapeutic applications, the heme peroxidase may be part of an enzyme-prodrug system. Enzyme-prodrug systems including heme peroxidases such as HRP are known in the art, particularly for cancer treatment (see, for example, Tupper et al. "In vivo characterization of horseradish peroxidase with indole-3-acetic acid and 5-bromoindole-3-acetic acid for gene therapy of cancer." Cancer Gene Therapy 17.6 (2010): 420-428). For example, targeted cancer treatment may include an enzyme-prodrug system including HRP with indole acetic acid (IAA), which oxidizes IAA to reduce the viability of cancer cells. Neither the above prodrugs IAA nor HRP are cytotoxic alone, indicating that it is necessary to combine these two substances in pure form, as well as in a form compatible and non-immunogenic to humans, to obtain the desired cytotoxic effect. Another study has shown that HRP can convert paracetamol into a potent cytotoxin (Tupper et al. "Use of horseradish peroxidase for gene-directed enzyme prodrug therapy with paracetamol." British Journal of Cancer 90.9 (2004): 1858-1862). However, the authors found that commercially available preparations of HRP from plants were not very effective in such conversion reactions, and therefore did not pursue this work further.

[0087] The heme peroxidase products produced by the present invention may find particular use in antibody directed enzyme prodrug therapy (ADEPT) (see, e.g., Bagshawe "Antibody-directed enzyme prodrug therapy (ADEPT) for cancer." Expert Review of Anticancer Therapy 6.10 (2006): 1421-1431). The principle of ADEPT is to localize an enzyme (such as HRP) to the tumor site, typically using an antibody directed to a tumor-associated antigen. A prodrug can be given to the patient that is converted to an active species in a site-specific manner. The heme peroxidase-encoding nucleic acid molecules may also find use in therapy, particularly gene therapy, such as gene-directed enzyme prodrug therapy (GDEPT).

[0088] In many applications, particularly industrial applications such as wastewater treatment (e.g., removal of chlorophenols), it may be advantageous when the heme peroxidase is immobilized on a solid support. Thus, it is preferred when the heme peroxidase product comprises a heme peroxidase immobilized on a solid support. Thus, the method of the present invention preferably further comprises the step of immobilizing the heme peroxidase or heme peroxidase conjugate on a solid support.

[0089] Among other advantages, enzyme immobilization allows for particularly high storage stability, improved reusability, and reduced operational process costs. Heme peroxidases immobilized on solid supports and their industrial applications are known in the art and are described, for example, in Tatsumi, et al. ("Removal of chlorophenols from wastewater by immobilized horseradish peroxidase." Biotechnology and Bioengineering 51.1 (1996): 126-130) and Sarno and Iuliano ("Immobilization of Horseradish Peroxidase on Fe 3 O 4 See, for example, Andreescu, et al. ("Chapter 7 - Nanostructured materials for enzyme immobilization and biosensors." The New Frontiers of Organic and Composite Nanotechnology. Elsevier, 2008, 355-394).

[0090] Nanomaterials are particularly suitable as solid supports due to their specific surface area and effective enzyme loading. For this embodiment, it is therefore preferred when the solid support is a nanoparticle. As used herein, a "nanoparticle" preferably has a surface area of ​​less than 1×10 in all three dimensions. -12 ~1×10 -6 m, and even more preferably in the range of 1×10 -9 ~1×10 -7 The nanoparticles are particles of any shape within the range of 100 nm. The nanoparticles are, for example, magnetite (Fe3 O 4 ) nanoparticles or gold nanoparticles. Magnetic nanoparticles are particularly preferred as they offer the additional advantage of facilitating separation by application of a magnetic field. Further preferred examples of solid supports are nanofibers, carbon / polyvinyl materials, carbon nanotubes, nanowires, nanorods, nanocrystals, mesoporous silica and composite materials.

[0091] In a further preferred embodiment, the solid support is a membrane, in particular a synthetic membrane, such as a polymeric membrane. Membranes containing immobilized heme peroxidase, methods for producing such membranes, and uses of such membranes (e.g., in wastewater treatment) are known in the art, for example from Vasileva et al. ("Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of phenol from water." International journal of biological macromolecules 44.2 (2009): 190-194).

[0092] In a further preferred embodiment, the heme peroxidase product is a kit. The method of the invention therefore preferably further comprises the step of packaging the heme peroxidase or the heme peroxidase conjugate in a kit. It is preferred when further components selected from buffers, reagents, instruction manuals are added to the kit. The heme peroxidase or the heme peroxidase conjugate may be provided in the kit in lyophilized form. In an alternative preferred embodiment, the heme peroxidase or the heme peroxidase conjugate is provided in a solution.

[0093] The heme peroxidases produced by the methods of the invention may further find use as reactants for coupled enzyme assays. In such assays, the enzyme of interest (e.g., glucose oxidase) is reacted with H 2 O 2 which can then be utilized by the heme peroxidase (see, e.g., Aumiller et al. "Coupled enzyme reactions performed in heterogeneous reaction media: experiments and modeling for glucose oxidase and horseradish peroxidase in a PEG / citrate aqueous two-phase system." The Journal of Physical Chemistry B 118.9 (2014): 2506-2517). Thus, in a preferred embodiment, H 2 O 2 An enzyme that produces

[0094] The heme peroxidase produced by the present invention may further be used as a reactant for polymer cross-linking. For example, HRP has been successfully used in the art for enzymatic cross-linking of dextran-tyramine conjugates for the preparation of hydrogels, e.g., as 3D scaffolds for cartilage tissue engineering applications (Jin et al. "Enzymatically crosslinked dextran-tyramine hydrogels as injectable scaffolds for cartilage tissue engineering." Tissue Engineering Part A 16.8 (2010): 2429-2440).

[0095] In order to facilitate understanding of the present invention, some terms are defined below. Terms defined herein have the meanings commonly understood by those skilled in the art in the context of the present invention. Terms such as "a", "an" and "the" are not intended to refer to a singular entity only, but include a general class of which a specific example may be used for illustrative purposes. Terms in this specification are used to describe specific embodiments of the present invention, but their use does not define the boundaries of the present invention except as outlined in the claims.

[0096] "Percent (%) amino acid sequence identity", "X% sequence identity" or "X% identical" (such as "70% sequence identity" or "70% identical") with respect to a reference polypeptide or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in said reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, using publicly available computer software, such as, for example, BLAST, BLAST-2, ALIGN, ALIGN-2, Megalign (DNASTAR), or the "needle" pairwise alignment application of the EMBOSS software package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are calculated using sequence alignments in the computer program "needle" of the EMBOSS software package (publicly available from the European Molecular Biology Laboratory; Rice et al., EMBOSS: the European Molecular Biology Open Software Suite, Trends Genet. 2000 Jun;16(6):276-7, PMID: 10827456).

[0097] The needle program can be accessed at the website http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / or downloaded for local installation as part of the EMBOSS package from http: / / emboss.sourceforge.net / . It runs on many widely used UNIX operating systems, such as Linux.

[0098] To align two protein sequences, the needle program is preferably run with the following parameters: Command line: needle -auto -stdout -asequence SEQUENCE_FILE_A -bsequence SEQUENCE_FILE_B -datafile EBLOSUM62 -gapopen 10.0 -gapextend 0.5 -endopen 10.0 -endextend 0.5 -aformat3 pair -sprotein1 -sprotein2(Align_format: pair Report_file:stdout)

[0099] The percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0100] Ratio (X / Y) x 100, where X is the number of amino acid residues scored as perfect matches by the sequence alignment program "needle" in the program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. If "the sequence of A is more than N% identical to the entire sequence of B," then Y is the entire sequence length of B (i.e., the total number of amino acid residues in B). Unless otherwise specifically stated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding section using the needle computer program.

[0101] Unless otherwise specified, all parameters used in this specification correspond to IUPAC SATP conditions (Standard Ambient Temperature and Pressure), in particular at a temperature of 25° C. and a pressure of 101.300 Pa.

[0102] Percentages (%) used herein correspond to weight / weight (w / w) or weight / volume (w / v) unless otherwise specified.

[0103] The present invention relates to the following preferred embodiments: Embodiment 1. A method for producing heme peroxidase from inclusion bodies (IBs), comprising: - providing said heme peroxidase in the form of IB; - solubilizing the IBs; - transferring the solubilized IBs into a refolding buffer to obtain a refolding mix; - adding a heme cofactor to the refolding mix; Including, The method, wherein the addition of the heme cofactor to the refolding mix is ​​distributed over a period of at least one hour.

[0104] Embodiment 2. The method of embodiment 1, wherein the addition of said heme cofactor to said refolding mix is ​​distributed over a period of at least 2 hours, preferably at least 3 hours, more preferably at least 6 hours, even more preferably at least 8 hours, in particular at least 10 hours.

[0105] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the heme cofactor is added to the refolding mix as a continuous feed.

[0106] Embodiment 4. The method according to any one of embodiments 1 to 3, wherein the heme cofactor is added to the refolding mix at a rate of 0.1 to 10 μmol / L / h, preferably 0.2 to 5 μmol / L / h, even more preferably 0.5 to 3 μmol / L / h, even more preferably 1 to 2.5 μmol / L / h, in particular 1.4 to 1.8 μmol / L / h.

[0107] Embodiment 5. The method according to any one of embodiments 1 to 4, wherein the refolding mix is ​​incubated for at least 1 hour, preferably at least 2 hours, even more preferably at least 4 hours, particularly at least 8 hours, prior to addition of the heme cofactor.

[0108] Embodiment 6. The method according to any one of embodiments 1 to 5, wherein at least 1 μmol / L, preferably at least 4 μmol / L, even more preferably at least 10 μmol / L, in particular at least 20 μmol / L of the heme cofactor is added to the refolding mix.

[0109] Embodiment 7. The method according to any one of embodiments 1 to 6, wherein 1 μmol / L to 200 μmol / L, preferably 4 μmol / L to 100 μmol / L, even more preferably 10 μmol / L to 50 μmol / L, in particular 15 μmol / L to 25 μmol / L of the heme cofactor is added to the refolding mix.

[0110] Embodiment 8. The method according to any one of embodiments 1 to 7, wherein at least 0.25 molar equivalents, preferably at least 0.5 molar equivalents, even more preferably at least 1 molar equivalent, in particular at least 2 molar equivalents of the heme cofactor relative to the heme peroxidase are added to the refolding mix.

[0111] Embodiment 9. The method according to any one of embodiments 1 to 8, wherein 0.25 to 10 molar equivalents, preferably 0.5 to 5 molar equivalents, even more preferably 1 to 4 molar equivalents, in particular 1 to 2 molar equivalents of the heme cofactor relative to the heme peroxidase are added to the refolding mix.

[0112] Embodiment 10. The method according to any one of embodiments 1 to 9, wherein the heme peroxidase is a class II or class III heme peroxidase, preferably a class III heme peroxidase.

[0113] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the heme peroxidase is horseradish peroxidase (HRP).

[0114] Embodiment 12. The method according to any one of embodiments 1 to 11, wherein said heme peroxidase does not contain a purification tag, in particular a polyhistidine tag.

[0115] Embodiment 13. The method according to any one of embodiments 1 to 12, wherein the heme peroxidase comprises an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, particularly at least 99% sequence identity to SEQ ID NO:3.

[0116] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the heme peroxidase comprises the amino acid sequence set forth in SEQ ID NO:3.

[0117] Embodiment 15. The method according to any one of embodiments 1 to 14, wherein the heme peroxidase consists of the amino acid sequence set forth in SEQ ID NO:4.

[0118] Embodiment 16. The method of any one of embodiments 1 to 15, wherein the heme cofactor is hemin.

[0119] Embodiment 17. The IB form of the heme peroxidase comprises: - culturing a host cell expressing the gene encoding said heme peroxidase; and - obtaining IBs from said host cells The method according to any one of embodiments 1 to 16, provided by

[0120] Embodiment 18 The method of embodiment 17, wherein the host cell is a prokaryotic cell, preferably an E. coli cell.

[0121] Embodiment 19. The method of embodiment 17 or embodiment 18, wherein the host cells are cultured in a volume of at least 2L, preferably at least 5L, more preferably at least 20L, even 50L, in particular at least 100L.

[0122] Embodiment 20. The method of any one of embodiments 1 to 19, wherein the solubilization comprises incubating the IBs in a solubilization buffer.

[0123] Embodiment 21. The method of embodiment 20, wherein said solubilization buffer has a pH of at least 8, preferably at least 8.5, more preferably at least 9.0, even more preferably at least 9.5.

[0124] Embodiment 22. The method of embodiment 20 or embodiment 21, wherein the solubilization buffer has a pH of 8 to 12.5, preferably 8.5 to 11.5, more preferably 9 to 11, even more preferably 9.5 to 10.5.

[0125] Embodiment 23. The method of any one of embodiments 20 to 22, wherein the solubilization buffer contains a reducing agent.

[0126] Embodiment 24. The method according to any one of embodiments 20 to 23, wherein the solubilization buffer has a redox state corresponding to a dithiothreitol (DTT) concentration of 1 to 50 mmol / L, preferably 2 to 25 mmol / L, more preferably 4 to 15 mmol / L, in particular 6 to 8 mmol / L.

[0127] Embodiment 25. The method according to any one of embodiments 20 to 24, wherein the solubilization buffer contains urea, preferably at a concentration of at least 4 mol / L, more preferably at least 5 mol / L, even more preferably at least 6 mol / L.

[0128] Embodiment 26. The method according to any one of embodiments 20 to 25, wherein the solubilization buffer contains 4 mol / L to 8 mol / L, preferably 5 mol / L to 7 mol / L, in particular 5.5 to 6.5 mol / L, of urea.

[0129] Embodiment 27. The method according to any one of embodiments 20 to 26, wherein the IBs are incubated in the solubilization buffer for at least 5 minutes, preferably at least 10 minutes, more preferably at least 15 minutes, in particular at least 30 minutes.

[0130] Embodiment 28. The method according to any one of embodiments 20 to 27, wherein the IBs are incubated in the solubilization buffer at a temperature between 4°C and 40°C, preferably between 10°C and 35°C, and even more preferably between 20°C and 25°C.

[0131] Embodiment 29. The method according to any one of embodiments 1 to 28, wherein the solubilized IBs are mixed with the refolding buffer in a ratio of 1:5 v / v to 1:250 (solubilized IBs:refolding buffer), preferably 1:10 to 1:100, even more preferably 1:20 to 1:80, in particular 1:25 to 1:60, and most preferably 1:30 to 1:50.

[0132] Embodiment 30. The method of any one of embodiments 1 to 29, wherein the refolding is carried out in a volume of at least 5 L, preferably at least 10 L, more preferably at least 25 L, even more preferably at least 100 L.

[0133] Embodiment 31. The method of any one of embodiments 1 to 30, wherein the refolding buffer has a pH of at least 8, preferably at least 8.5, more preferably at least 9, even more preferably at least 9.5.

[0134] Embodiment 32. The method according to any one of embodiments 1 to 31, wherein the refolding buffer has a pH of 8 to 12.5, preferably 8.5 to 11.5, more preferably 9 to 11, and even more preferably 9.5 to 10.5.

[0135] Embodiment 33. The method according to any one of embodiments 1 to 32, wherein the pH difference between the solubilization buffer and the refolding buffer is a pH increment of less than 2, preferably a pH increment of less than 1, more preferably a pH increment of less than 0.5, even more preferably a pH increment of less than 0.2, in particular a pH increment of less than 0.1.

[0136] Embodiment 34. The method of any one of embodiments 1 to 33, wherein the refolding buffer contains an oxidizing agent.

[0137] Embodiment 35. The method according to any one of embodiments 1 to 34, wherein the refolding buffer has a redox state corresponding to a glutathione disulfide (GSSG) concentration of 0.2 to 6 mM GSSG, preferably 0.4 to 4 mM GSSG, more preferably 0.8 to 2.5 mM GSSG, particularly 1.1 to 1.6 mM GSSG.

[0138] Embodiment 36. The method according to any one of embodiments 1 to 35, wherein the refolding buffer contains 0.5 to 3 mol / L, preferably 1 mol / L to 2.7 mol / L, even more preferably 1.5 mol / L to 2.4 mol / L, in particular 1.8 to 2.2 mol / L of urea.

[0139] Embodiment 37. The method according to any one of embodiments 1 to 36, wherein the molar ratio between DTT and GSSG in the refolding mix is ​​from 1:2 (DTT:GSSG) to 1:30, preferably from 1:3 to 1:20, even more preferably from 1:4 to 1:15, in particular from 1:6 to 1:8.

[0140] Embodiment 38. The method according to any one of embodiments 1 to 37, wherein the refolding mix has a reduction potential of -105 mV to 135 mV, preferably -90 mV to 105 mV, even more preferably -80 mV to 80 mV, even more preferably -70 mV to 60 mV, and most preferably -60 mV to 50 mV.

[0141] Embodiment 39. The method of any one of embodiments 1 to 38, further comprising adding salt to the refolding mix.

[0142] Embodiment 40. The method according to embodiment 39, wherein the salt is added to the refolding mix to an ionic strength of 0.5 to 6 mol / L, preferably 1.5 to 5 mol / L, even more preferably 2.5 to 4.5 mol / L, in particular 3.5 to 4.5 mol / L.

[0143] Embodiment 41. The method according to embodiment 39 or embodiment 40, wherein the salt is sodium chloride, preferably at a concentration of 0.5 to 6 mol / L, more preferably 1.5 to 5.5 mol / L, even more preferably 2.5 to 5 mol / L, in particular 3.5 to 4.5 mol / L.

[0144] Embodiment 42. The method according to any one of embodiments 39 to 41, wherein the salt is ammonium sulfate, preferably at a concentration of 0.25 to 1.5 mol / L, preferably 0.5 to 1.4 mol / L, even more preferably 0.8 to 1.2 mol / L.

[0145] Embodiment 43. The method according to any one of embodiments 39 to 42, wherein the salt has a salting-out strength, as determined by its position in the Hofmeister series, between that of ammonium sulfate and sodium chloride, and preferably, the concentration of the salt is 1.25 to 6 mol / L, more preferably 0.5 to 5 mol / L, even more preferably 1.0 to 4.5 mol / L, and most preferably 1.5 to 4 mol / L.

[0146] Embodiment 44. The method according to any one of embodiments 39 to 43, wherein the salt has a salting-out strength, as determined by its position in the Hofmeister series, weaker than sodium chloride, and preferably the concentration of the salt is at least 2 mol / L, preferably at least 4 mol / L, more preferably at least 4.5 mol / L.

[0147] Embodiment 45. The method according to embodiment 44, wherein the concentration of the salt is from 2 mol / L to 12 mol / L, preferably from 4 mol / L to 10 mol / L, and even more preferably from 4.5 mol / L to 8 mol / L.

[0148] Embodiment 46. The method according to any one of embodiments 1 to 45, further comprising a centrifugation step to remove impurities from the refolded heme peroxidase.

[0149] Embodiment 47. The method according to any one of embodiments 1 to 46, further comprising a filtration step to remove impurities from the refolded heme peroxidase.

[0150] Embodiment 48. The method according to any one of embodiments 1 to 47, further comprising purifying the refolded heme peroxidase.

[0151] Embodiment 49 The method of embodiment 48, wherein the heme peroxidase is purified after the heme cofactor is added to the refolding mix.

[0152] Embodiment 50 The method of embodiment 48 or embodiment 49, wherein the heme peroxidase is purified by chromatography.

[0153] Embodiment 51. The method of any one of embodiments 48 to 50, wherein the heme peroxidase is purified by hydrophobic interaction chromatography (HIC).

[0154] Embodiment 52. The method of embodiment 51, wherein the stationary phase is a butyl sepharose resin.

[0155] Embodiment 53. A method for producing a heme peroxidase product, comprising producing heme peroxidase by a method according to any one of embodiments 1 to 52.

[0156] Embodiment 54 The method of embodiment 53, further comprising activating the heme peroxidase for the conjugation reaction.

[0157] Embodiment 55 The method according to embodiment 53 or embodiment 54, further comprising a conjugation reaction to obtain a heme peroxidase conjugate.

[0158] Embodiment 56 The method of embodiment 55, wherein said heme peroxidase is conjugated to a binding agent, preferably an antibody.

[0159] Embodiment 57. The method of embodiment 55, wherein said heme peroxidase is conjugated to an antibody fragment, preferably a single chain variable fragment (scFv) or an antigen-binding fragment (Fab).

[0160] Embodiment 58. The method of embodiment 55, wherein the heme peroxidase is conjugated to an antibody mimic, preferably an antibody mimic selected from the group consisting of adnectins, affibodies, anticalins, DARPins, engineered Kunitz-type inhibitors, and monobodies.

[0161] Embodiment 59. The method of embodiment 55, wherein the heme peroxidase is conjugated to an antibody-binding protein, preferably Protein A, Protein G, or Protein L.

[0162] Embodiment 60 The method of embodiment 55, wherein said heme peroxidase is conjugated to a further protein, preferably streptavidin.

[0163] Embodiment 61. The method of any one of embodiments 53 to 60, further comprising purifying the heme peroxidase or the heme peroxidase conjugate.

[0164] Embodiment 62. The method of any one of embodiments 53 to 61, further comprising freezing the heme peroxidase or the heme peroxidase conjugate.

[0165] Embodiment 63. The method of any one of embodiments 53 to 62, further comprising lyophilizing the heme peroxidase or the heme peroxidase conjugate.

[0166] Embodiment 64. The method of any one of embodiments 53 to 63, further comprising immobilizing the heme peroxidase or heme peroxidase conjugate on a solid support.

[0167] Embodiment 65. The method of embodiment 64, wherein the solid support is a nanoparticle, preferably a magnetic nanoparticle or nanofiber.

[0168] Embodiment 66 The method of embodiment 64, wherein the solid support is a carbon / polyvinyl material.

[0169] Embodiment 67. The method of embodiment 64, wherein the solid support is a membrane.

[0170] Embodiment 68. The method of any one of embodiments 53 to 67, further comprising packaging the heme peroxidase or the heme peroxidase conjugate into a kit.

[0171] Embodiment 69 The method of embodiment 68, wherein further components selected from buffers, reagents, instruction manuals are added to the kit.

[0172] Embodiment 70.H 2 O 2 70. The method of embodiment 69, wherein an enzyme producing the

[0173] The present invention is further illustrated by the following figures and examples, but is not limited thereto. [Brief description of the drawings]

[0174] [Figure 1A] Effect of different heme cofactor concentrations and different addition times to the refolding mix on the volumetric activity and refolding yield, respectively. [Figure 1B] Response contour plot (DoE4) of volumetric activity [U / mL] as a response for different heme cofactor addition times and heme cofactor concentrations as factors. [Diagram 2] Response contour plot for volumetric activity [U / mL] depending on the DTT concentration in the solubilization mix and the GSSG concentration in the refolding buffer (DoE1). [Diagram 3] Response contour plots at pH 8.5, pH 9.25 and pH 10 with different DTT and GSSG concentrations (both in mM) for DoE3. [Figure 4] Redox potential and volumetric activity (at-line sampling) in reactor run 2. 20 h after the start of refolding, 20 μM hemin was added, at which point the redox signal shows a sharp increase. [Diagram 5] Redox potential and volumetric activity in reactor run 5. Prior to the end of the hemin feed, samples were withdrawn every 2 hours and incubated at a final hemin concentration of 20 μM before measurement (circles). After the start of the hemin feed, samples were withdrawn every 2 hours and volumetric activity was measured immediately (squares). [Figure 6] Elution of active HRP and impurities on HIC1 using a step gradient. Active HRP elutes between 120 and 125 mL (75% Buffer B), while hydrophobic impurities elute starting at 131 mL (100% Buffer B). [Figure 7] Elution of active HRP and impurities on HIC2 using a linear gradient. Active HRP elutes between 125 mL and 135 mL, whereas hydrophobic impurities elute starting at 145 mL. [Figure 8]Elution of active HRP and impurities on HIC3 using a linear gradient. Active HRP elutes late in the gradient (near 90% buffer B) and overlaps with the elution of hydrophobic impurities that elute at 100% buffer B. [Figure 9] Elution of active HRP and impurities on HIC4 using a step gradient. Although separation of active HRP from hydrophobic impurities is possible using 90% buffer B, this leads to strong tailing of the active HRP peak, resulting in a trade-off between lower concentrations of active HRP and poorer recovery. [Figure 10] Elution of active HRP and impurities on HIC5 using a step gradient. Active HRP elutes between 1250 and 1400 mL (75% Buffer B), while hydrophobic impurities begin eluting at 1400 mL (100% Buffer B). [Figure 11] Chromatogram of HIC run with refolding at pH 8.5. Pure HRP elutes at 75% Buffer B, while hydrophobic impurities elute at 100% Buffer B. [Figure 12] Chromatogram of HIC run with refolding at pH 10. Pure HRP elutes at 75% Buffer B, while hydrophobic impurities elute at 100% Buffer B. [Figure 13] Residual activity of HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (mHRP, SEQ ID NO: 4) compared to recombinant wild-type HRP (rHRP, SEQ ID NO: 2) and plant-derived HRP (pHRP). 150 μl of pHRP (triangles), mHRP (squares) and rHRP (circles) at a concentration of 2.9 μM were incubated at 60° C. for up to 10 hours in 50 mM BisTris / HCl (pH 7), 0.5 M NaCl, 7% glycerol. Initial and residual enzyme activity was measured by ABTS as described in Example 1 and plotted as % against incubation time. mHRP showed more than 13-fold improved thermostability over rHRP and 1.7-fold improved thermostability over the plant-derived (i.e. glycosylated) pHRP. [Figure 14] Enzyme activity obtained from 3 different refolding experiments. "Batch addition": the heme cofactor was added in a batch after 20 h refolding time (first column). "Hemin feed 10 h": the heme cofactor was added as a 10 h feed 8 h after the start of refolding (second column). "Hemin feed 1 h": the heme cofactor was added as a 1 h feed 8 h after the start of refolding; measurements were taken immediately after the end of the feed (last column) and after a further incubation of 10 h (third column). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0175] Example 1. Materials and Methods (chemical substance) Oxidized L-glutathione (GSSG) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were from AppliChem. Hemin was purchased from Sigma (bovine hemin, ≥90%). Dithiothreitol (DTT) and all other chemicals were from Roth.

[0176] Bacterial strains and growth conditions The hrp gene encoding HRP variant C1a was codon-optimized for E. coli and obtained from Genscript USA (Piscataway, NJ, USA). Plasmid pET21d+ was used for HRP inclusion body production in the cytoplasm. A stop codon was introduced so that the protein was produced without any tag. HRP was produced in E. coli BL21(DE3) in a 10L Biostat Cplus stainless steel bioreactor (Satorius, Germany). HRP production was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 12 h in fed-batch culture using DeLisa medium. Biomass was harvested by centrifugation and wet biomass was stored at -20°C until further processing.

[0177] (Homogenization and washing) The biomass was resuspended in 3-5 mL of buffer A / g wet biomass (buffer A: 50 mM TRIS / HCl; pH 8; 500 mM NaCl; 1.5 mM EDTA) using an IKA T10 Basic ULTRA-TURRAX and homogenized (>1300 bar, 3 passes, chilled) (using a GEA Niro Soavi Panda PLUS). The homogenized suspension was centrifuged (15650 g; 20 min, 4° C.), the supernatant was discarded and the cell debris was resuspended in 10 mL of buffer B / g wet cell debris (buffer B: 50 mM TRIS / HCl; pH 8; 500 mM NaCl; 2 M urea) and centrifuged again (15650 g; 20 min, 4° C.). The washing step with buffer B was repeated once. The IBs / cell debris were then resuspended in water (5 mL water / g wet cell debris) and the suspension was aliquoted into pre-weighed 50 mL reaction tubes, centrifuged (15650 g; 20 min, 4° C.) and the pellets were stored at −20° C. until further use.

[0178] (Solubilization) For solubilization, an aliquot of frozen IBs was thawed, weighed to calculate wet inclusion body (wIB) weight, and resuspended in the appropriate solubilization buffer (Solubilization Buffer 1: 50 mM TRIS / HCl; pH 8; 6 M urea / Solubilization Buffer 2: 50 mM glycine; pH 10; 6 M urea) to a wIB concentration of 100 g / L. After resuspension, DTT was added (1 M DTT stock solution was used) to give a final concentration of between 1 mM and 28.44 mM DTT in the solubilization mix, which was incubated (room temperature; 0.5 h; gentle agitation) and subsequently centrifuged (20379 g; 20 min; 4 °C). The supernatant was used immediately for refolding and the pellet was discarded.

[0179] Example 2. Small-scale refolding experiments (Analysis method) (Bradford): Protein concentration was determined using the Bradford assay by measuring absorbance at 595 nm using a Genesys20 Photometer (Thermo Scientific) after 10 min of incubation at room temperature, with bovine serum albumin used as standard.

[0180] (Enzyme activity measurement): HRP enzyme activity was measured by Tecan Infinite M200PRO using flat-bottom polystyrene 96-well plates. Depending on the concentration of correctly folded HRP, samples were diluted 1:50 to 1:200 in dilution buffer (dilution buffer: 20 mM Bis-Tris (pH 7); 7% v / v glycerol). 170 μL of ABTS solution (50 mM KH 2 PO 4 Mix 10 μL of diluted sample with 5 mM ABTS in 100 mM NaCl (pH 5) and then add 20 μL of HO. 2 O 2 (10 mM H 2 O 2 The reaction was started by the addition of 100 mM NaCl. Immediately after, the change in absorbance at 420 nm was recorded (at 30° C.) over a period of 2 min. The volumetric enzyme activity was calculated using the following formula:

[0181]

number

[0182] V total Total volume in the cuvette [μl] ΔA / min: change in absorbance [ΔAbsorption (420 nm) / min] dilution: Sample dilution V sample ...sample volume [μl] d: length of the beam path through the reaction (d = 0.58 cm) ε...Extinction coefficient (ε 420 =36mM -1 cm -1 ).

[0183] (SEC-HPLC): The purity of the active HRP fraction after the HIC capture step was determined using SEC-HPLC. For this, an XBridge Protein BEH SEC column, 200 Å, 3.5 μm, 7.8 mm×150 mm (Waters) was used. The method was carried out at 0.5 mL / min for 18 min with 100% Buffer A (Buffer A: 80 mM phosphate (pH 6.8); 250 mM KCl). The column was kept at a constant temperature of 25° C. and measurements were taken at 214 nm, 280 nm and 404 nm.

[0184] (Reinheitszahl (RZ)): Reinheitszahl was measured as the ratio of absorbance at 404 nm to that at 280 nm. Absorbance measurements were performed using a Hitachi Double Beam Spectrophotometer U-2900.

[0185] (MODDE10): Design of Experiments (DoE) was designed and analyzed using Umetrics MODDE10.

[0186] (Experimental Setup) General materials and methods were as described in Example 1. All small-scale refolding experiments were performed using 2 mL reaction tubes. The solubilizate was diluted 1:40 with the appropriate refolding buffer (Refolding buffer 1: 20 mM TRIS / HCl; pH 8.5; 2 M urea; 2 mM CaCl 2 7% v / v glycerol; varying GSSG concentrations / Refolding buffer 2: 20 mM glycine; pH 10; 2 M urea; 2 mM CaCl 2 ; 7% v / v glycerol; varying GSSG concentrations), followed by incubation with gentle agitation for 48 h at 4° C. A 1 mM hemin stock solution used for all experiments was prepared in 100 mM KOH.

[0187] (DoE1): In the first DoE, the DTT concentration for solubilization (solubilization buffer 1; pH 8) and the GSSG concentration in the refolding buffer (refolding buffer 1; pH 8.5) were varied (see Table 1), while the hemin addition was kept constant at 20 μM after 20 h. A central composite face-centered design (CCF) was used with the volume activity after refolding as the response.

[0188] [Table 1]

[0189] (DoE2): In the second DoE, the DTT concentration for solubilization (solubilization buffer 1; pH 8), the GSSG concentration in the refolding buffer (refolding buffer 1; pH 8.5), and the protein concentration in the refolding mix were varied (see Table 2), and the hemin addition was kept constant at 20 μM after 20 h. A CCF (central composite design) was used with volumetric activity and specific activity after refolding as responses.

[0190] [Table 2]

[0191] (DoE3): In the third DoE, the DTT concentration for solubilization, the GSSG concentration in the refolding buffer, and the pH of the solubilization and refolding buffers were varied (see Table 3). At pH 8.5, solubilization and refolding buffer 1 was used, and at pH 10, solubilization and refolding buffer 2 was used. Hemin addition was kept constant at 20 μM after 20 hours. A CCF (central composite design) was used with volume activity after refolding as the response.

[0192] [Table 3]

[0193] (DoE4): In this DoE, the time and concentration of hemin addition were varied between 0 h and 24 h after the start of refolding, and between 6 μM and 80 μM hemin, respectively. The exact factors used are shown in Table 4. Solubilization buffer 1 (pH 8) and refolding buffer 1 (pH 8.5) were used in all experiments. Volume activity was used as the response to optimize both factors.

[0194] [Table 4]

[0195] (Result: addition of hemin) To evaluate the optimal concentration of hemin and the optimal addition time after the start of refolding, several different conditions were screened in small-scale experiments using volume activity as the response. The results are presented in Figure 1. With hemin added at 0 hours after the start of refolding, the refolding yield showed a strong dependence on hemin concentration, with higher hemin concentrations leading to a more than 10-fold reduction compared to the optimized conditions. Moreover, the refolding yield is reduced by 60% even at the lowest hemin concentration (6 μM). This effect is reduced with later hemin addition times, with little effect of hemin concentration seen after 24 hours of addition. 20 μM was chosen for further experiments.

[0196] (Result: Redox system) In a first step, a design of experiments was carried out using DTT and GSSG concentrations to obtain the optimal redox system during solubilization and refolding (DoE1). This approach has the advantage of covering two unit operations, solubilization and refolding, and using the refolding yield (volume activity) as the response. This maximizes the final yield of the process compared to using the solubilization yield as the response, for example, to optimize a single unit operation. Figure 2 shows the contour plot for the carried out DoE1, where the maximum refolding yield is obtained with 7.11 mM DTT (buffer 1; pH 8) and 1.27 mM GSSG (buffer 1; pH 8.5) during solubilization, as calculated using the MODDE optimization function. With a dilution of 1:40 used (solubilization mix in refolding buffer), this is equivalent to a GSSG:DTT ratio of 7:1.

[0197] A DoE similar to DoE1 was performed, except that pH (7; 8.5; 10) was added as an additional factor to cover the pH dependence of redox versus DTT / GSSG. Refolding at pH 7 gives lower results for all combinations of DTT and GSSG, which greatly influences the model. This leads to poor predictions for DTT and GSSG at pH 8.5 and 10. Therefore, pH 7 was removed from the model. Figure 3 shows the 4D contour plot for the final model using volumetric activity as response. At pH 10, the highest yield is achieved with 6.7 mM DTT and 1.26 mM GSSG.

[0198] Example 3. Refolding reactor experiments General materials and methods were as described in Example 1 unless otherwise specified. Analytical methods were used as described in Example 2.

[0199] (Reactor settings) For larger scale refolding, an Infors Labfors5 with a vessel volume of 3.6 L was used. All data collection and process control was performed using a Lucullus PIMS. Temperature was kept constant at 10°C during refolding using a Lauda Alpha R8 thermostat connected to the double jacket of the glass vessel. Temperature was monitored using a temperature sensor connected to the Infors Labfors5. In addition, pH, dO2 and redox potential were monitored. The pH and dO2 were measured by respective probes connected to the Infors Labfors5. The redox potential was monitored using a Hamilton EasyFerm Plus ORP Arc425 connected to a Lucullus process system. Hemin feed was applied using a LAMBDA PRECIFLOW peristaltic liquid pump combined with a Sartorius Entris scale, allowing PID-feedforward control using Lucullus.

[0200] The final refolding volume of the vessel was kept constant at 1.2 L (resulting in the use of 30 mL of lysate and a dilution of 1:40). The same buffer compositions as mentioned for the small-scale experiments (Example 2; Refold Buffer 1 or Refold Buffer 2) were used.

[0201] (Reactor Run) (Reactor Run 1): In this run, the solubilization mix contained 1 mM DTT and the refolding buffer contained 0.35 mM GSSG. Solubilization buffer 1 (pH 8) and refolding buffer 1 (pH 8.5) were used. Hemin was added 20 hours after the start of refolding to a final concentration of 20 μM.

[0202] (Reactor run 2): In this run, the solubilization mix contained 7.11 mM DTT and the refolding buffer contained 1.27 mM GSSG. Solubilization buffer 1 (pH 8) and refolding buffer 1 (pH 8.5) were used. Hemin was added 20 hours after the start of refolding to a final concentration of 20 μM. Samples (2 mL in reaction tubes) were taken every 2 hours and hemin was added to a final concentration of 20 μM (only for samples taken before hemin addition; samples after hemin addition already contained 20 μM), incubated (2 hours; 4° C., gentle agitation) and then enzyme activity was measured.

[0203] (Reactor Run 3): In this run, the solubilization mix contained 1 mM DTT and the refolding buffer contained 0.35 mM GSSG. Solubilization buffer 2 (pH 10) and refolding buffer 2 (pH 10) were used. Hemin was added 20 hours after the start of refolding to a final concentration of 20 μM.

[0204] (Reactor Run 4): In this run, the solubilization mix contained 7.11 mM DTT and the refolding buffer contained 1.27 mM GSSG. Solubilization buffer 2 (pH 10) and refolding buffer 2 (pH 10) were used. Hemin was added 20 hours after the start of refolding to a final concentration of 20 μM.

[0205] (Reactor run 5): In this run, the solubilization mix contained 7.11 mM DTT and the refolding buffer contained 1.27 mM GSSG. Solubilization buffer 1 (pH 8) and refolding buffer 1 (pH 8.5) were used. A constant feed (2 mL, 1 mM hemin / h; final concentration: 20 μM hemin) was applied from 8 hours to 20 hours after the start of refolding (12 hours feed time). For reactor 2, samples were withdrawn every 2 hours and activity was measured. After the start of the hemin feed, samples were measured both directly (at low hemin concentration at the start of the hemin feed) and after hemin addition to a final hemin concentration of 20 μM and incubation for 2 hours.

[0206] (Result: Hemin addition time) Based on the small-scale optimization (Example 2), a final concentration of 20 μM hemin added 20 hours after the start of refolding was used for reactor run 2. Before hemin addition (i.e., the first 20 hours), samples were taken every 2 hours and the activity was measured at-line (Figure 4). To measure the volumetric activity, which correlates with the success of refolding, 20 μM hemin was added to each sample immediately after sampling and the activity was measured after 2 hours of incubation. After hemin addition to the reactor (i.e., after 20 hours), samples were further taken every 2 hours but no further hemin was added. Furthermore, the samples were incubated under the same conditions as above (2 hours before measurement). As can be seen for the at-line activity measurements, refolding is complete after approximately 10 hours and then the activity plateaus until hemin is added. The increase in activity after hemin addition to the reactor is probably due to better conditions in the reactor, such as a better surface-to-volume ratio and better mixing.

[0207] [Table 5]

[0208] These experiments surprisingly demonstrate that it is advantageous to incubate the refolding mix for a period of time prior to the addition of the heme cofactor. The hydrophobic nature of hemin appears to have promoted aggregation during the refolding process. As shown using small-scale experiments, higher concentrations of hemin significantly reduced the refolding yield when added early, an effect that was not present when added late. Adding a hydrophobic substance (in this case hemin) early in the reaction may have promoted protein aggregation, leading to lower refolding yields. However, once the folding reaction is complete (approximately 10 hours for HRP under the conditions used), the addition of hemin does not lead to aggregation (for all concentrations tested) since hemin incorporation is the last step in holoenzyme formation.

[0209] (Result: Linear hemin feed) In reactor run 5, a linear hemin feed was applied 8 hours after the start of refolding, with a total feed duration of 12 hours. Samples were drawn (every 2 hours) according to the sampling procedure described for reactor run 2. After the start of the hemin feed, samples were measured both once without further hemin addition (i.e., at the low hemin concentration at the start of the hemin feed; samples b1-b10 in Table 6) and once after hemin was added to a final hemin concentration of 20 μM and incubated for an additional 2 hours (samples a1-a9 in Table 6).

[0210] [Table 6]

[0211] The beneficial effect of hemin addition distributed over a certain period of time versus a single batch addition is evident from a comparison of reactor runs 2 and 5. Run 2, sample 4 and run 5, sample a4 are both based on a batch addition of 20 μM hemin 6 hours after the start of refolding and measurement after another 2 hours. As a result, the observed volumetric activities are very similar (31.97 ± 0.52 vs. 33.10 ± 1.49), demonstrating that the sample results can be compared between these runs. The difference between batch addition and addition over a longer period of time is evident from a comparison of run 2, sample 10 (batch addition of 20 μM hemin after 18 hours, measured after 20 hours: 35.44 ± 0.86) and run 5, sample b6 (20 μM hemin added as a linear feed from 8 to 20 hours after refolding, measured after 20.5 hours: 62.40 ± 2.10). The only difference between these two samples was the duration of hemin addition (all at once as a batch or distributed over 12 hours), clearly demonstrating that distributing the hemin addition over a longer period leads to superior results.

[0212] Figure 5 shows the redox potential and the measured volumetric activity. Compared to reactor run 2 (Figure 4), the beginning of the refolding is very similar in both runs, with the volumetric activity achieved after 8 hours being 32.0 in run 2 and 33.1 in run 5. However, the linear hemin feed applied in run 5 leads to a 25% increase in the refolding yield compared to run 2 after hemin addition.

[0213] (Result: Redox system) Four reactor experiments were performed to confirm the results obtained using the small-scale experiment (Example 2). The conditions and final specific activities are listed in Table 7. Overall, these results are in good agreement with those obtained using the small-scale experiment, with the optimized DTT / GSSG concentration at pH 10 showing the highest refolding yields.

[0214] [Table 7]

[0215] Example 4. Capture after refolding General materials and methods were as described in Example 1. Analytical methods were used as described in Example 2.

[0216] In this example, hydrophobic interaction chromatography (HIC) was used as a capture step after refolding. This has the advantage that the binding conditions require high salt concentrations that precipitate impurities such as excess hemin, aggregates and impurities, while correctly folded HRP is stable up to high salt concentrations. This has the advantage that these impurities are separated from the load used for the capture step, resulting in higher binding capacity and easier washing and regeneration of the chromatography resin.

[0217] (Small-scale precipitation experiments) To find the best salt concentration for the capture step using HIC (hydrophobic interaction chromatography), different concentrations of (NH 4 ) 2 SO 4 and NaCl were tested in 2 mL reaction tubes. The appropriate amount of salt (see Table 8) was weighed in a 2 mL reaction tube and 1 mL of refold mix was added (after refolding was completed). The reaction tube was mixed to dissolve the salt and incubated at room temperature for 30 minutes with weak stirring. After centrifugation (20379 g; 20 min; 4° C.), the supernatant was used for Bradford and activity measurements. The best conditions were then used to prepare the load for the various HIC experiments using larger volumes. Therefore, the salt was added slowly under continuous stirring within 10 minutes, and then the solution was incubated at room temperature for 20 minutes with stirring and then centrifuged (20379 g; 20 min; 22° C.).

[0218] [Table 8]

[0219] An AKTA Pure system (GE Healthcare) was used for all chromatographic steps described. Three wavelengths (214 nm, 280 nm and 404 nm) and conductivity were monitored.

[0220] Two different salts, namely (NH 4 ) 2 SO 4 and NaCl were tested for the precipitation step. To find the optimal salt concentration, several salt concentrations of both salts were tested in 2 mL reaction tubes. The results are listed in Tables 9 and 10. Usually, the lowest salt concentration required for the target protein to bind in the HIC resin is used for the preparation of the load, since this concentration results in the lowest amount of impurities binding. However, here, the highest amount that does not precipitate active HRP was chosen. For one, this ensured that a large amount of impurities was precipitated (see purification factor in Table 9). On the other hand, almost all impurities that bound to the resin in these conditions were more hydrophobic and therefore could also bind at lower salt concentrations. In addition, the difference in binding strength made it easier to separate active HRP from these impurities. For this reason, 1M (NH 4 ) 2 SO 4 Or 4M NaCl was chosen for the load preparation.

[0221] [Table 9]

[0222] [Table 10]

[0223] The results obtained for the small-scale experiments were verified using bench-scale experiments with a volume of 150 mL, as listed in Table 11. The volumetric activity recoveries for selected salt concentrations were calculated as (NH 4 )2 SO 4 The specific activities were 96% and 95% for NaCl and NaCl. 4 ) 2 SO 4 The concentrations of β-aminobutyric acid and β-aminobutyric acid increased by 2.5-fold and 4.5-fold in NaCl, respectively, providing favorable conditions for loading in the subsequent HIC step.

[0224] [Table 11]

[0225] (HIC Experiment 1 (HIC1)) The load was prepared as described above by adding 267 g NaCl / 1 L of refolding mix. 1 mL HiTrap Butyl FF (GE Healthcare) was used at a flow rate of 0.5 mL / min (75 cm / h; 0.5 CV / min; CV = column volume). The column was equilibrated with Buffer A (Buffer A: 20 mM Bis-Tris pH 7; 7% glycerol; 4 M NaCl) and a 49 mL load was applied after all signals were constant during equilibration. After these loads, a wash step with Buffer A (8 mL; 8 CV) was performed. Then, step elutions were performed with 25% Buffer B (Buffer B: 20 ​​mM Bis-Tris pH 7; 7% glycerol / 8 mL; 8 CV), 75% Buffer B (10 mL; 10 CV) and 100% (17 mL; 17 CV) Buffer B, and active HRP was eluted in 75% Buffer B. Volumetric enzyme activity [U / mL] and protein concentration were measured for all fractions. Purity of the active pool was determined using SEC-HPLC and Reinheitszahl.

[0226] This capture step represents an optimized method for purification and capture of HRP after refolding. Figure 6 shows the step elution used in this experiment, and Table 12 shows the corresponding analytical results. Active HRP was concentrated about 9-fold to 0.5 g / L for the chromatography step, which is expected to improve further upon scale-up. More hydrophobic impurities were eluted at 100% buffer B, leading to good resolution for this method. The ratio of 404 nm signal to 280 nm signal suggests high purity for the active fractions, which was further confirmed by SEC-HPLC (purity ≧98%). Thus, this represents a method to obtain pure, correctly folded, and fully active HRP produced from E. coli inclusion bodies.

[0227] [Table 12]

[0228] (HIC Experiment 2 (HIC2)) 132g of (NH 4 ) 2 SO 4 The load was prepared as described above by adding 1 mL of refold mix per 1 L. A 1 mL HiTrap Octyl FF (GE Healthcare) was used at a flow rate of 1 mL / min (150 cm / hr; 1 CV / min). The column was equilibrated with Buffer A (Buffer A: 20 mM Tris (pH 8.5); 7% glycerol; 1 M (NH 4 ) 2 SO 4), and a 50 mL load was applied after all signals had become constant during equilibration. After these loads, a wash step with buffer A (16 mL; 16 CV) was performed. Then, a linear gradient elution was performed with 0-100% B (buffer B: 20 ​​mM Bis-Tris (pH 7); 7% glycerol) in 30 mL (30 min; 30 CV). Volumetric enzyme activity [U / mL] was measured for all fractions. By this point, the highest percentage of active HRP was found in the flow-through, so no further analysis was applied.

[0229] The capture step used HiTrap Octyl FF (1 mL) (GE Healthcare), a resin with low hydrophobicity. 4 ) 2 SO 4 ), 75% of the active HRP did not bind to the column and was found in the flow-through (see FIG. 7 and Table 13). This could be improved by using higher salt concentrations, but in small-scale experiments, 1M (NH 4 ) 2 SO 4 It was shown that at higher salt concentrations than 0.1, the recovery decreased. It was therefore concluded that the resin is less favorable for the capture step of active HRP after refolding. Since the hydrophobicity of the column depends on the density of the bound ligand, this could be improved if an octyl resin with a higher ligand density were used. The ligand density for Octyl Sepharose 4 Fast Flow (5 μmol / mL resin) is about 10 times lower than that of Butyl Sepharose 4 Fast Flow (50 μmol / mL resin) used in HIC1.

[0230] [Table 13]

[0231] (HIC experiments 3 and 4 (HIC3 and HIC4)) For HIC experiment 3, the load was prepared as described above by adding 267 g NaCl / 1 L of refolding mix. 1 mL of HiTrap Phenyl FF (High Sub) (GE Healthcare) was used at a flow rate of 1 mL / min (150 cm / h; 1 CV / min). The column was equilibrated with buffer A (buffer A: 20 mM Tris (pH 8.5); 7% glycerol; 4 M NaCl) and a 50 mL load was applied after all signals were constant during equilibration. After these loads, a wash step with buffer A (9 mL; 9 CV) was performed. Then, a linear gradient elution was performed from 0 to 100% B in 30 mL (buffer B: 20 ​​mM Bis-Tris (pH 7); 7% glycerol) (30 min; 30 CV). Volumetric enzyme activity [U / mL] was measured for all fractions. Fractions containing active HRP showed lower concentrations and did not show good separation from the impurities, so further analysis was not applied.

[0232] For HIC run 4, the load was prepared as described above by adding 267 g NaCl / 1 L refolding mix. 1 mL HiTrap Phenyl FF (High Sub) (GE Healthcare) was used at a flow rate of 0.5 mL / min (75 cm / h; 0.5 CV / min). The column was equilibrated with Buffer A (Buffer A: 20 mM Tris (pH 8.5); 7% glycerol; 4 M NaCl) and a 50 mL load was applied after all signals were constant during equilibration. These loads were followed by a wash step with Buffer A (10 mL; 10 CV). Then, step elution was performed with 60% buffer B (buffer B: 20 ​​mM Bis-Tris (pH 7); 7% glycerol / 9 mL; 9 CV), 90% buffer B (26 mL; 26 CV) and 100% (32 mL; 32 CV) buffer B, with active HRP eluted with 90% buffer B. Volumetric enzyme activity [U / mL] was measured for all fractions. Fractions containing active HRP showed lower concentrations, indicating no good separation from impurities, so further analysis was not applied.

[0233] In HIC3 and HIC4, a resin with higher hydrophobicity than HIC1 was used (HiTrap Phenyl FF(High Sub)). In HIC3, gradient elution was used but resulted in poorer separation of active HRP from the more hydrophobic impurities as shown in Figure 8. Due to the high hydrophobicity of the resin, both fractions eluted very close to each other, with active HRP near 90% Buffer B and impurities at 100% Buffer B. Therefore, step elution was tested in HIC4 shown in Figure 9. While the separation improved, the elution volume for the active HRP fraction eluting at 90% Buffer B was also increased as it showed strong tailing.

[0234] (HIC experiment 5 (HIC5)) The finished refolding mix from reactor run 4 (example 3) was used as material for this capture step. The pH was adjusted to 8.5 under stirring with 2M HCl. The load was then prepared as described above by adding 267 g NaCl / 1 L refolding mix. A column packed with Butyl Sepharose 4 Fast Flow (GE Healthcare) and with a volume of 80 ml was used at a flow rate of 8 mL / min (90 cm / h). The column was equilibrated with Buffer A (Buffer A: 20 mM Bis-Tris (pH 7); 4 M NaCl) and a load of 751 mL was applied after all signals were constant during equilibration. This load was followed by a washing step with 20% Buffer B (Buffer B: 20 ​​mM Bis-Tris (pH 7) / 1.5 CV). Then, a step elution was performed with 75% buffer B (3 CV) and 100% (3 CV) buffer B, and active HRP was eluted in 75% buffer B. Volumetric enzyme activity [U / mL] and protein concentration were measured for all fractions. Purity of the active pool was determined using a Reinheitszahl.

[0235] To confirm the suitability of refolding at pH 10 with a subsequent capture step, the refold mix from reactor run 4 was used as the load for the HIC. The pH of the refold mix was adjusted to 8.5 before salting out to prevent any changes in binding behavior. Figure 10 shows the elution of the active HRP fraction and impurities. Active HRP was concentrated to a final concentration of 0.95 g / L and a final volume activity of 1500 U / mL, with a Reinheitszahl comparable to that of plant HRP (2.72). These results confirmed the prediction that scale-up from 1 mL column volume would lead to higher concentrations, as discussed in HIC1. Furthermore, the capture step discussed in HIC1 was shown to be scalable and applicable to the refolding process at pH 10, since the pH could be adjusted to 8.5 without loss of active HRP. Thus, a capture step using 4 M NaCl for salting out and subsequent hydrophobic interaction chromatography proved to be particularly advantageous.

[0236] Example 5. Overall process (Analysis method) The analytical method was used as described in Example 2 with the following differences / additions. (Protein concentration): Protein concentration was determined using the method by Bradford (Bradford, MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 1976. 72(1-2): p. 248-254). 200 μl of Bradford solution was mixed with 5 μl of sample and the change in absorbance at 595 nm was measured over 10 min using a Tecan Infinite M200 PRO instrument.

[0237] (Reversed Phase HPLC): HRP concentration in samples was measured by RP HPLC using a PolyPhenyl BioResolve-RP-mAb (2.7 μm, 3.0×100 mm) column. The method was run for 10 min with the following program: Flow rate was 1.2 ml / min, 25% B for 0.5 min, 55% B in a linear gradient for 8 min, 55% B for 0.5 min, and then 25% B for 1 min (A=MilliQ water with 0.1% trifluoroacetic acid (TFA); B=acetonitrile with 0.1% TFA). The column was kept at a constant temperature of 75° C. and measurements were taken at 214 nm, 280 nm, and 404 nm.

[0238] (Size Exclusion HPLC): The purity of the active HRP fraction after the HIC capture step was determined using SEC-HPLC. For this, a BEH200A SEC 1.7 μm, 4.6×300 mm, 3.5 μm (Waters) column was used. The method was run for 18 min at 0.3 mL / min with 100% Buffer A (Buffer A: 80 mM phosphate (pH 6.8); 250 mM KCl). The column was kept at a constant temperature of 30° C. and measurements were taken at 214 nm, 280 nm and 404 nm.

[0239] (Process Run) The cell lines and growth conditions, as well as homogenization and inclusion body washing, were performed as described in Example 1. The refolding reactor was used as described in Example 3.

[0240] For solubilization, an aliquot of frozen IB was thawed, weighed to calculate wet inclusion body (wIB) weight, and resuspended in the appropriate solubilization buffer (Solubilization Buffer 1: 50 mM TRIS / HCl; pH 8.5; 6 M urea / Solubilization Buffer 2: 50 mM glycine; pH 10; 6 M urea) to a wIB concentration of 100 g / L (3.5 g IB = 35 ml solubilization mix (resulting in 30 ml solubilization mix after centrifugation). After resuspension, DTT was added (1 M DTT stock solution was used) to a final concentration of 7.11 mM DTT in the solubilization mix, and the solubilization mix was incubated (4°C; 0.5 h; gentle agitation) and subsequently centrifuged (20379 g; 20 min; 4°C). The supernatant was used immediately for refolding and the pellet was discarded.

[0241] Reactor runs were performed at pH 8.5 and pH 10 as follows.

[0242] At pH 8.5, the final refolding volume in the vessel was 1.2 L (resulting from the use of 30 mL of lysate and a dilution of 1:40). The refolding buffer was 20 mM TRIS / HCl (pH 8.5), 2 M urea, 7% glycerol, 2 mM CaCl 2 , containing 1.27 mM GSSG. A constant feed (2.4 mL, 1 mM hemin / h; final concentration 20 μM hemin) was applied from 8 h initiation of refolding until 18 h later (10 h feed time), and the reactor run was terminated after 19 h. For salting out, 0.27 g NaCl / ml refolding mix was added over 30 min with stirring at room temperature. The refolding mix was then centrifuged at 17,568 g, 4 °C for 30 min in a Thermo Scientific LYNX Sorvall 6000 centrifuge. The supernatant was used as the load for the HIC (approximately 1250-1300 ml after salt addition) and the pellet was discarded.

[0243] At pH 10, the final vessel refolding volume was 1.2 L (resulting from the use of 30 mL of lysate and a dilution of 1:40). The refolding buffer was 20 mM glycine, pH 10 (adjusted with HCl), 2 M urea, 7% glycerol, 2 mM CaCl 2 , containing 1.27 mM GSSG. A constant feed (2.4 mL, 1 mM hemin / h; final concentration 20 μM hemin) was applied from 8 h to 18 h after the start of refolding (10 h feed time) and the reactor run was terminated after 19 h. Prior to salting out, the pH was lowered from pH 10 to pH 8.5 with HCl. 0.27 g NaCl / ml refolding mix was then added over 30 min with stirring at room temperature. The refolding mix was then centrifuged at 17,568 g for 30 min at 4 °C in a Thermo Scientific LYNX Sorvall 6000 centrifuge. The supernatant was used as the load for the HIC (approximately 1250-1300 ml after salt addition) and the pellet was discarded.

[0244] For HIC, a column with a bed volume of 80 ml packed with Butyl Sepharose 4 Fast Flow (GE Healthcare) was used. The column was equilibrated with Buffer A (Buffer A: 20 mM Bis-Tris pH 7; 4 M NaCl) at a flow rate of 113 cm / h until all signals were constant. Then, loads of 1250-1300 mL were applied at a flow rate of 90 cm / h. After these loads, a washing step with 20% Buffer B (Buffer B: 20 ​​mM Bis-Tris pH 7) was performed for 2 CV at a flow rate of 90 cm / h. Then, step elution was performed with 75% Buffer B (79 cm / h) and 100% (90 cm / h) Buffer B, and active HRP was eluted in 75% Buffer B. Volumetric enzyme activity [U / mL] and protein concentration were measured for all fractions. Purity of the active pool was determined using a Reinheitszahl.

[0245] (result) Method development experiments performed on a small scale found that pH 10 is more suitable for refolding than pH 8.5 (see Example 2). Therefore, two reactor runs were performed with the same amount of HRP wet inclusion bodies, one for refolding at pH 8.5 and one for refolding at pH 10. A summary of the results can be seen in Table 14. Compared to the refolding yield at pH 8.5 (44%), refolding at pH 10 led to a strongly increased refolding yield (74%). Also, the Reinheitszahl is higher and the total yield of pure HRP increases by 1.7-fold at pH 10. This reconfirms the results of the small scale DoE (Example 2). Figures 11 and 12 show the chromatograms of the HIC runs. Here, it can be seen that refolding at pH 10 leads to a significant reduction of hydrophobic impurities and a large and sharp HRP peak at 75% of buffer B.

[0246] [Table 14]

[0247] Example 6. Materials and Methods for HRP Mutation Studies (Expression and purification of HRP mutants) Plant HRP type VI-A (catalog number: P6782) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All HRP variants produced in E. coli consisted of the sequence set forth in SEQ ID NO:2 with the indicated mutations unless otherwise specified.

[0248] Expression hosts and plasmids Standard molecular cleaning techniques were performed as previously described (Humer and Spadiut. "Improving the performance of horseradish peroxidase by site-directed mutagenesis." International Journal of Molecular Sciences 20.4 (2019): 916). The hrp gene encoding HRP variant C1A (wild-type HRP; SEQ ID NO:2) was codon-optimized for E. coli and obtained from GenSript USA Inc. (Piscataway, NJ, USA). HRP was produced from pSF-T7-LacO-NH2-dsbA(OG4591) (Oxford Genetics Ltd., Oxford, UK) or pET21d+ (Novagen, San Diego, CA, USA) in E. coli strain BL21(DE3) (Lucigen, Middleton, WI, USA). Plasmid pSFT7 encodes a Dsb tag for export into the periplasm, which is cleaved after translocation. The plasmid pET21d+ was used for the production of HRP inclusion bodies in the cytoplasm, and a stop codon was introduced so that the protein was produced without any tag.

[0249] Protein expression and purification from inclusion bodies (IBs) BL21(DE3) cells carrying the vector pET21d+ carrying the hrp gene (or its variants) without any N- or C-terminal tags were cultured in SB medium (32 gL -1 Tryptone; 20gL -1 Yeast extract; 5gL -1 NaCl; 5 mM NaOH) was used. Ampicillin was 100 mg L -1 The preculture was added to 50 mL of SB Amp The medium was grown overnight at 37°C with shaking (250 rpm) and inoculated into 2.5 L Ultra Yield Flasks (UYF) to a final volume of 500 mL SB Amp The medium was incubated at an optical density (OD 600The cells were cultured at an OD of 0.5. 600 The cells were grown at 37°C with shaking (250 rpm) until 37°C, after which hrp expression was induced by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After 20 h of growth at 25°C and 250 rpm, the cells were harvested by centrifugation (5000g, 20 min, 4°C).

[0250] The biomass was resuspended in 3-5 mL of buffer A / g wet biomass (buffer A: 50 mM TRIS / HCl; pH 8; 500 mM NaCl; 1.5 mM EDTA) using an IKA T10 Basic ULTRA-TURRAX and homogenized (using a GEA Niro Soavi Panda PLUS) (>1300 bar, 3 passes, refrigerated). The homogenized suspension was centrifuged (15650 g; 20 min, 4 °C), the supernatant was discarded and the cell debris was resuspended in 10 mL of buffer B / g wet cell debris (buffer B: 50 mM TRIS / HCl; pH 8; 500 mM NaCl; 2 M urea) and centrifuged again (15650 g; 20 min, 4 °C). The washing step with buffer B was repeated once. The IBs / cell debris were then resuspended in water (5 mL water / g wet cell debris) and the suspension was aliquoted into pre-weighed 50 mL reaction tubes, centrifuged (15650 g; 20 min, 4° C.) and the pellets were stored at −20° C. until further use.

[0251] For solubilization, an aliquot of frozen IB was thawed, weighed to calculate wet inclusion body (wIB) weight, and resuspended in the appropriate solubilization buffer (50 mM TRIS / HCl; pH 8.5; 6 M urea) to a wIB concentration of 100 g / L. After resuspension, DTT was added (1 M DTT stock solution was used) to a final concentration of 7.11 mM DTT in the solubilization mix, and the solubilization mix was incubated (4° C.; 0.5 h; gentle agitation) and subsequently centrifuged (20379 g; 20 min; 4° C.). The supernatant was used immediately for refolding and the pellet was discarded.

[0252] Resuspend the lysate in an appropriate refolding buffer (e.g., 20 mM TRIS / HCl (pH 8.5), 2 M urea, 7% glycerol, 2 mM CaCl 2 The solution was diluted 1:40 in 10 mM NaCl (1.27 mM GSSG) and hemin was added to a final concentration of 20 μM, and refolding was carried out at 10° C. for 19 hours.

[0253] The protein was further purified by hydrophobic interaction chromatography (HIC). A column with a bed volume of 80 ml packed with Butyl Sepharose 4 Fast Flow (GE Healthcare) was used at 8 mL / min (90 cm / h). The column was equilibrated with Buffer A (Buffer A: 20 mM Bis-Tris pH 7; 4 M NaCl) at a flow rate of 113 cm / h until all signals were constant. Then, 1250-1300 mL loads were applied at a flow rate of 90 cm / h. After these loads, a washing step with 20% Buffer B (Buffer B: 20 ​​mM Bis-Tris pH 7) was performed for 2 CV at a flow rate of 90 cm / h. Then, a step elution was performed with 75% Buffer B (79 cm / h) and 100% (90 cm / h) Buffer B, and active HRP was eluted in 75% Buffer B.

[0254] For some variants, protein production and purification was instead carried out using the most preferred method described in Example 5 (pH 10).

[0255] (Reaction rate parameters) Enzyme kinetic parameters were determined for the substrates ABTS, TMB and hydrogen peroxide in a 96-well plate assay using a Tecan Infinite M200 PRO instrument (Tecan, Manndorf, Switzerland).

[0256] For the assay using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate, the reaction mixture in each well of a 96-well plate contained a saturating hydrogen peroxide concentration of 1 mM and varying TMB concentrations (20-550 μM) in a final volume of 200 μL of 50 mM phosphate-citrate buffer (pH 5). Protein samples (5 μL) were mixed with 175 μL of the TMB-buffer mixture and the reaction was initiated with 20 μL of hydrogen peroxide solution (10 mM). The increase in absorbance was followed at 652 nm for 60 s at 30 °C in a Tecan Infinite M200PRO instrument. Reaction kinetic parameters were analyzed using Sigma Plot software (Systat Software INC., San Jose, CA, USA) and ε 652 Extinction coefficient = 39 mM -1 cm -1 (See Josephy, et al. "The horseradish peroxidase-catalyzed oxidation of 3,5,3',5'-tetramethylbenzidine. Free radical and charge-transfer complex intermediates." Journal of Biological Chemistry 257.7 (1982): 3669-3675.)

[0257] For measurements using ABTS as substrate, the reaction mixture in each well of a 96-well plate contained a saturating hydrogen peroxide concentration of 1 mM and varying ABTS concentrations (0.1-7 mM) in a final volume of 200 μL of 50 mM phosphate-citrate buffer (pH 5). Protein samples (5 μL) were mixed with 175 μL of the ABTS-buffer mixture and the reaction was initiated with 20 μL of hydrogen peroxide solution (10 mM). The increase in absorbance was followed at 420 nm for 120 s at 30 °C in a Tecan Infinite M200PRO instrument. Kinetic parameters were analyzed using Sigma Plot software (Systat Software INC., San Jose, CA, USA) and ε420 Extinction coefficient = 36 mM -1 cm -1 (See Childs and Bardsley. "The steady-state kinetics of peroxidase with 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen." Biochemical Journal 145.1 (1975): 93-103.)

[0258] For measurements using hydrogen peroxide as substrate, the reaction mixture in each well of a 96-well plate contained 10 mM saturating ABTS and varying hydrogen peroxide concentrations (0.001-1 mM) in a final volume of 200 μL of 50 mM phosphate-citrate buffer (pH 5). Protein samples (5 μL) were mixed with 145 μL of hydrogen peroxide-buffer mixture and the reaction was initiated with 50 μL of ABTS solution (40 mM). The increase in absorbance was followed at 420 nm for 120 s at 30 °C in a Tecan Infinite M200PRO instrument. Kinetic parameters were analyzed using Sigma Plot software (Systat Software INC., San Jose, CA, USA) and ε 420 Extinction coefficient = 36 mM -1 cm -1 (See Childs and Bardsley. "The steady-state kinetics of peroxidase with 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen." Biochemical Journal 145.1 (1975): 93-103.)

[0259] (thermal stability) The thermal stability of the enzyme variants was evaluated in 50 mM BisTris / HCl (pH 7), 7% glycerol, 500 mM NaCl at 60° C. Enzyme activity with ABTS was measured for HRP wild type (SEQ ID NO:2) and HRP N13D / N57S / N255D / N268D after 0, 30, 60, 90, and 120 minutes; variants HRP N13D / N57S / N175S / N255D / N268D, HRP N13D / N57S / P146Q / N175S / N255D / N268D, HRP N13D / N57S / N175S / N255D / N268D, and HRP N275K after 0, 90, 180, 300, 420, and 588 minutes. N13D / N57S / P146Q / N175S / N255D / N268D / N275K; and plant HRP after 0, 90, 180, 300 and 420 min. The enzyme concentration of all variants, including plant HRP, was 2.86 μM upon heat treatment. The samples were then cooled on ice for 5 min and then centrifuged at 16162 g for 15 min at 4° C. The residual activity was then measured with 7 mM ABTS using a Tecan Infinite M200PRO instrument. The reaction mixture contained 5 μL of protein, a saturating hydrogen peroxide concentration of 1 mM, and 7 mM ABTS in a total volume of 200 μl of 50 mM phosphate-citrate buffer (pH 5). The increase in absorbance was followed at 420 nm for 120 s at 30° C. Residual enzyme activity was plotted against incubation time and the half-life at 60° C. was calculated using the rate of inactivation in the following formula:

[0260] t 1 / 2 =ln(2) / k in In the formula, t 1 / 2 is the half-life, and k in is the slope of the log residual activity.

[0261] Example 7. Thermal stability of HRP mutants Several mutants of HRP were expressed and purified and the thermostability was measured as described in Example 6. Many mutations were found to increase thermostability. The most preferred mutant HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4) showed more than 13-fold improved thermostability over wild-type HRP (SEQ ID NO: 2) and 1.7-fold improved thermostability even over the plant-derived (i.e., glycosylated) enzyme. Importantly, significantly higher thermal stability was observed for the combination of mutations P146Q and N275K (HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K) than either the mutations P146Q (HRP N13D / N57S / P146Q / N175S / N255D / N268D) or N275K (HRP N13D / N57S / N175S / N255D / N268D / N275K) alone.

[0262] [Table 15]

[0263] Example 8. Kinetic parameters of HRP variants Several mutants of HRP were expressed and purified, and kinetic parameters were determined for the substrates ABTS, TMB, and hydrogen peroxide as described in Example 6. HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4) was found to be significantly more active than HRP N13D / N57S / N175S / N255D / N268D with the substrates TMB and hydrogen peroxide. Mutations P146Q and N275K were found to have a strong beneficial effect on enzyme activity.

[0264] [Table 16]

[0265] [Table 17]

[0266] [Table 18]

[0267] Example 9. Comparison between plant-derived HRP and wild-type HRP The kinetic parameters and thermostability of HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4) were compared to those of wild-type HRP (SEQ ID NO: 2) and plant-derived HRP (pHRP). The kinetic parameters and thermostability were determined as described in Example 6.

[0268] The thermal stability measurements are shown in Figure 13. As already described in Example 7 herein above, the most preferred mutant HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (mHRP, SEQ ID NO: 4) showed a more than 13-fold improved thermal stability over wild-type HRP (rHRP, SEQ ID NO: 2) and a further 1.7-fold improved thermal stability over plant-derived (i.e. glycosylated) pHRP (see Table 15). It was also found that, despite this strongly improved thermal stability, a similar catalytic effect was observed as both wild-type HRP (rHRP, SEQ ID NO: 2) and plant-derived HRP (see Tables 19 and 20 below).

[0269] [Table 19]

[0270] [Table 20]

[0271] Example 10. Site-saturation mutagenesis at positions 146 and 275 To test the effect of all possible amino acid exchanges at positions P146 and N275, site-saturation mutagenesis of these positions was performed. As a starting point for the site-saturation mutagenesis, the preferred mutant HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 3) was selected. The effect of the amino acid exchanges at positions 146 and 275 was examined individually.

[0272] (Library generation) The following plasmids were constructed by standard molecular cloning techniques: Whole-plasmid PCR of HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 3) in pSF-T7 was used to introduce mutations into the hrp gene at positions 146 and 275 by site-saturation mutagenesis. A 6.3 kb fragment was amplified with the respective oligonucleotides to generate a site-saturation library (Table 21). All oligonucleotides were purchased from Microsynth (Balgach, Switzerland). The oligonucleotides were phosphorylated by the following protocol: 300 pmol primer DNA, 1x T4PNK buffer (NEB), 1 mM ATP, 5% PEG, 10 units T4 polynucleotide kinase (PNK, NEB). The reaction was incubated at 37°C for 45 min and then heat-inactivated at 65°C for 20 min. Each PCR reaction contained 1x Q5 reaction buffer, 200μM dNTP Mix, 200nM of both forward and reverse phosphorylated primers, 100ng of template vector DNA, and 1U Q5 High-Fidelity DNA polymerase. PCR products were purified by Monarch PCR & DNA Cleanup Kit from New England Biolabs (NEB, Ipswich, MA, USA) and template plasmid DNA was removed by FastDigest DpnI (Thermo Scientific™, Waltham, MA, USA) digestion. 2FDU (FastDigest units) of DpnI were added to the cloned PCR products and incubated at 37°C for 4 hours. After heat inactivation at 80°C for 20 minutes, the plasmid was blunt-end ligated: 50ng of plasmid DNA, 1x T4 DNA ligase buffer (NEB), 400 sticky end units T4 DNA ligase (NEB), overnight at 16°C. After heat inactivation at 65° C. for 20 min, the plasmid was transformed into BL21(DE3).

[0273] [Table 21]

[0274] (screening) Positively transformed cells were harvested from the selection plate and transferred to a 96-well plate in 200 μl of SB medium (32 gL) in a plastic box to prevent drying. -1 Tryptone; 20gL -1 Yeast extract; 5gL -1 NaCl; 5 mM NaOH; 50 mg L -1 The plates were grown with 10 μl of the master plate in 10 ml of SB medium (100 μl of kanamycin) for 16 hours at 37° C. and 250 rpm. Afterwards, 90 μl of 75% glycerol was added to the master plate, after which they were stored at −80° C. 10 μl of the master plate was inoculated onto a slave plate containing 190 μl of SB medium, which was now supplemented with 2 mM CaCl 2 in a final volume of 200 μl. 2 6 μM hemin, and 0.1 mM IPTG. The cells were grown in a plastic box at 25° C. and 250 rpm for 16 h, and cell density was determined by measuring absorbance at 595 nm in a Tecan Infinite M200PRO (Tecan, Mandorf, Switzerland) plate reader. The plates were then centrifuged at 5000 g for 6 min at 4° C. in a Thermo-Fisher Lynx Sorvall centrifuge, and the cells were diluted with 5 Uml -1 of DNaseI and 1 / 2 protease inhibitor cocktail tablets (Complete tablets, EDTA-free; Roche Diagnostics GmbH, Mannheim, Germany) and 200 mM MgCl 2The cells were thoroughly resuspended in 200 μl / well of B-PER Bacterial Protein Extraction Reagent (Thermo Scientific, Waltham, MA, USA) using a 5000 g / well buffer. Cell lysis was performed at room temperature for 15 min, followed by centrifugation at 5000 g for 20 min at 4°C. Total protein content was then measured by the Bradford method. 90 μl of each well was transferred to a new plate and incubated at 80°C for 20 min (position 146) or at 80°C for 15 min (position 275), after which both the heated and control plates were centrifuged at 5000 g for 20 min at 4°C. Then, the cells were incubated in a total volume of 200 μl with 395 μM TMB (position 146) or 406 μM TMB (position 275), 1 mM H in 50 mM phosphate-citrate buffer (pH 5). 2 O 2 Enzyme activity was measured using 10 μl of cell lysate and 10 μl of lysate. Measurements were performed at 30° C. and the increase in absorbance at 652 nm (ε=3.9×10 for blue TMB radical) was 4 M -1 cm -1 ) was monitored for 120 seconds by a Tecan Infinite M200 PRO plate reader. Initial and residual activities were normalized using total protein concentration, and thermostability was expressed as the percentage of residual activity relative to initial activity. The procedure was the same as described above. 180 colonies were screened for each position, corresponding to an expected library completeness of >99%. Each plate contained 6 colonies of HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (corresponding to SEQ ID NO: 3) as a positive control.

[0275] (result) The results for selected mutants are shown below in Table 22 (position P146) and Table 23 (position N275). Because this example uses a 96-well plate-based screening assay, the variability (standard deviation) is higher than with other assays reported herein. Therefore, the results for each mutant should only be compared within each plate measured.

[0276] As can be seen from Tables 22 and 23, several different amino acid exchanges at positions 146 and 275 were found to lead to excellent thermostability. Several amino acid exchanges at each position gave advantageous results. For position 146, 146A, 146R, 146V, 146E, and especially 146Q were found to be particularly advantageous (all within the standard deviation of the most preferred variant 146Q). For position 275, the best results were observed for 275R, 275D, 275S, 275Q, 275A, 275E, and especially 275K (all within the standard deviation of the most preferred variant 275K).

[0277] [Table 22]

[0278] [Table 23]

[0279] Example 11. Distribution of heme cofactor addition over various periods of time (method) The process runs were performed as described in Example 5 with the following differences: cell lines and growth conditions, as well as homogenization and inclusion body washes, were performed as described in Example 1. The refolding reactor was used as described in Example 3.

[0280] For solubilization, an aliquot of frozen IB was thawed, weighed to calculate the wet inclusion body (wIB) weight, and resuspended in the appropriate solubilization buffer (solubilization buffer 2: 50 mM glycine; pH 10; 6 M urea) to a wIB concentration of 100 g / L (3.5 g IB = 35 ml solubilization mix (resulting in 30 ml solubilization mix after centrifugation)). After resuspension, DTT was added to a final concentration of 7.11 mM DTT in the solubilization mix, and the solubilization mix was incubated (4°C; 0.5 h; gentle agitation) and subsequently centrifuged (20379 g; 20 min; 4°C). The supernatant was used immediately for refolding and the pellet was discarded. The final refolding volume of the vessel was 1.2 L (resulting in the use of 30 mL of lysate and a dilution of 1:40). The refolding buffer was 20 mM glycine (pH 10) (adjusted with HCl), 2 M urea, 7% glycerol, 2 mM CaCl 2 , containing 1.27 mM GSSG.

[0281] Three different hemin addition regimes were compared: (A) 20 hours of refolding followed by batch addition of hemin to a final concentration of 20 μM and an additional 5 hours of incubation before measurement; (B) 8 hours of refolding followed by 10 hours of hemin feed addition (2.4 ml of 1 mM hemin / hour; final concentration 20 μM hemin) and an additional 1 hour of incubation before measurement; and (C) 8 hours of refolding followed by 1 hour of hemin feed addition (24 ml of 1 mM hemin / hour; final concentration 20 μM hemin) and an additional 10 hours of incubation. In experiment (C), samples were taken and measured immediately after the end of the feed and further measurements were made after an additional 10 hours of incubation.

[0282] Protein concentration and enzyme activity were determined as described in Example 2.

[0283] (result) The results obtained are shown in FIG. 14. A clear benefit was found in distributing the heme cofactor addition over a 1 hour period. Even when measured immediately after the end of the 1 hour hemin feed (i.e. after a total refolding time of 9 hours), the specific activity was found to be 1.4 times higher than in the batch addition experiment (wherein the total refolding time was 25 hours). When samples from the 1 hour feed experiment were measured after an additional incubation of 10 hours (i.e. after a total refolding time of 19 hours), the specific activity was 1.7 times higher than that from the batch addition. Distributing the hemin addition over a longer period of 10 hours (experiment (B); 19 hours total refolding time) led to an increase in specific activity of as much as 2-fold over the batch addition.

[0284] Example 12. Kinetic parameters and thermostability of additional HRP variants The beneficial effects of the mutations at positions P146 and N275 were investigated for wild-type HRP (SEQ ID NO: 2), HRP N175S and HRP N13D / N57S / N175S / N255D / N268D. Furthermore, the beneficial effects of the single mutants and their combinations were investigated. In this regard, the role of the mutations at positions P146 and N275 on the biochemical properties was determined by measuring the specific enzyme activity and thermostability at 60°C.

[0285] Materials and Methods The following mutants of wild-type HRP (SEQ ID NO: 2) were created and verified by Sanger sequencing: HRP P146Q, HRP N175S, HRP N275K, HRP P146Q / N275K, HRP P146Q / N175S, HRP N175S / N275K, HRP P146Q / N175S / N275K. Purification was performed as described in Example 6. Kinetic parameters and thermostability were also determined as described in Example 6.

[0286] (Specific enzyme activity (ABTS)) The specific activity (units / mg protein) of all HRP variants was tested with the substrate ABTS in a Tecan plate reader (Table 24). HRP P146Q showed 1.4-fold higher specific activity than HRP wild type. Interestingly, HRP N175S showed lower specific activity; however, HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4) was able to attenuate this activity reduction, restoring it to the value of HRP wild type.

[0287] [Table 24]

[0288] (Specific enzyme activity (H 2 O 2 )) In addition, the specific activity (units / mg protein) was determined using hydrogen peroxide substrate. Here, the same trend was observed in the data for ABTS, with the variant P146Q leading to an increase in specific activity and the introduction of N175S leading to lower values. This was also the case for the double mutants P146Q / N175S and N175S / N275K, as well as the triple mutant P146Q / N175S / N275K. When N175S was absent or when the additional mutation of SEQ ID NO:4 was present, the specific activity was similar to or even better than SEQ ID NO:2.

[0289] [Table 25]

[0290] (Specific enzyme activity (TMB)) For the substrate TMB, the differences in specific activity between the HRP variants were less pronounced, but P146Q again showed a significant increase in specific enzyme activity, while N175S showed the lowest U / mg compared to the wild type.

[0291] [Table 26]

[0292] (thermal stability) The enzyme stability at 60°C was investigated for all HRP variants, and surprisingly, N175S was not the only factor responsible for the increased stability at high temperature. HRP N13D / N57S / N255D / N268D increased the enzyme half-life by 1.5-fold, while HRP N175S improved the stability by 7.7-fold, and their combination increased the stability by 13-fold compared to HRP wild type, suggesting a synergistic effect (Table 27). The fact that HRP N13D / N57S / N175S / N255D / N268D and HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4) showed similar stability may lead us to speculate that the quadruple mutant combined with N175S is responsible for this improvement.

[0293] For P146Q and N275K, the stability of the single mutant and the double mutant P146Q / N275K was slightly reduced compared to the HRP wild type. However, the triple mutant P146Q / N175S / N275K showed the same stability as N175S alone, while the double mutants P146Q / N175S and N175S / N275K were more unstable. This suggests that only one of the mutants in combination with N175S shows an unfavorable effect on stability, which is mitigated by the combination, suggesting a synergistic effect between P146Q, N275K, and N175S. The same effect is found for HRP N13D / N57S / P146Q / N175S / N255D / N268D and HRP N13D / N57S / N175S / N255D / N268D / N275K when compared to HRP N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4).

[0294] [Table 27]

[0295] (Conclusion) It was found that the substitution P146Q itself led to a strong increase in HRP enzyme activity for all substrates tested, for example with the substrate ABTS, which reached a 1.4-fold increase over the wild-type enzyme (SEQ ID NO: 2) and a 2-fold higher specific activity compared to HRP N175S.

[0296] The substitution N175S was found to strongly increase the thermal stability of HRP. This effect was observed on N175S by itself and even more strongly in combination with the mutated N-glycosylation site amino acids N13D / N57S / N255D / N268D, and a synergistic effect was observed. Combination of the single mutation P146Q or the single mutation N275K with N175S led to a slight decrease in stability. However, this decrease was alleviated when both P146Q and N275K were combined with N175S, suggesting a synergistic effect between P146Q, N275K, and N175S.

[0297] N175S was found to reduce the enzyme activity in some substrates. This effect was, however, attenuated by N13D / N57S / P146Q / N175S / N255D / N268D / N275K (SEQ ID NO: 4). Therefore, this mutant offers a combination of high thermostability and optimal kinetic performance. (Additional Note) The present disclosure includes the following aspects. Section 1: 1. A method for producing heme peroxidase from inclusion bodies (IBs), comprising: - providing said heme peroxidase in the form of IB; - solubilizing the IBs; - transferring the solubilized IBs into a refolding buffer to obtain a refolding mix; - adding a heme cofactor to the refolding mix; Including, The method, wherein the addition of the heme cofactor to the refolding mix is ​​distributed over a period of at least 1 hour, preferably at least 2 hours, more preferably at least 3 hours, even more preferably at least 6 hours, in particular at least 10 hours. Section 2: 2. The method of claim 1, wherein the heme cofactor is added to the refolding mix as a continuous feed. Section 3: 3. The method of claim 1 or 2, wherein the refolding mix is ​​incubated for at least 1 hour, preferably at least 2 hours, even more preferably at least 4 hours, especially at least 8 hours prior to addition of the heme cofactor. Section 4: Item 4. The method according to any one of Items 1 to 3, wherein the heme peroxidase is a class II or class III heme peroxidase, preferably a class III heme peroxidase, in particular horseradish peroxidase (HRP). Section 5: The method according to any one of Items 1 to 4, wherein the heme peroxidase comprises an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% sequence identity to SEQ ID NO: 3, in particular, the heme peroxidase comprises the amino acid sequence set forth in SEQ ID NO: 3, and most preferably, the heme peroxidase consists of the amino acid sequence set forth in SEQ ID NO: 4. Item 6: Item 6. The method according to any one of Items 1 to 5, wherein the heme cofactor is hemin. Section 7: The IB form of heme peroxidase is: - culturing a host cell expressing the gene encoding said heme peroxidase; and - obtaining IBs from said host cells, 7. The method according to any one of items 1 to 6, wherein the host cell is a prokaryotic cell, preferably an E. coli cell. Section 8: Item 8. The method according to any one of items 1 to 7, wherein the solubilization comprises incubating the IBs in a solubilization buffer, preferably the solubilization buffer has a pH of 8 to 12.5, preferably 8.5 to 11.5, more preferably 9 to 11, even more preferably 9.5 to 10.5. Section 9: Item 9. The method according to Item 8, wherein the solubilization buffer has a reducing state corresponding to a dithiothreitol (DTT) concentration of 1 to 50 mmol / L, preferably 2 to 25 mmol / L, more preferably 4 to 15 mmol / L, particularly preferably 6 to 8 mmol / L. Section 10: Item 10. The method according to any one of items 1 to 9, further comprising a step of purifying the refolded heme peroxidase after the heme cofactor is added to the refolding mix, preferably wherein the method further comprises a centrifugation step to remove impurities from the refolded heme peroxidase, and / or the heme peroxidase is purified by hydrophobic interaction chromatography (HIC). Section 11: Item 11. A method for producing a heme peroxidase product, comprising producing heme peroxidase by the method according to any one of items 1 to 10. Section 12: 12. The method according to claim 11, further comprising a conjugation reaction to obtain a heme peroxidase conjugate, preferably wherein the heme peroxidase is conjugated to a binding agent, preferably wherein the heme peroxidase is conjugated to an antibody, an antibody fragment, an antibody mimetic, an antibody binding protein, or streptavidin. Section 13: Item 13. The method of item 11 or 12, further comprising purifying, freezing, and / or lyophilizing the heme peroxidase or the heme peroxidase conjugate. Section 14: Item 14. The method according to any one of Items 11 to 13, further comprising a step of immobilizing the heme peroxidase or heme peroxidase conjugate on a solid support, preferably the solid support being a nanoparticle, a carbon / polyvinyl material, or a membrane. Section 15: Item 15. The method according to any one of Items 11 to 14, further comprising packaging the heme peroxidase or the heme peroxidase conjugate into a kit.

Claims

1. 1. A method for producing heme peroxidase from inclusion bodies (IB), comprising: - providing said heme peroxidase in the form of IBs; - solubilizing said IBs; - transferring the solubilized IBs into a refolding buffer to obtain a refolding mix; - adding a heme cofactor to the refolding mix; Including, The method, wherein the addition of the heme cofactor to the refolding mix is ​​distributed over a period of at least one hour.

2. 2. The method of claim 1, wherein the heme cofactor is added to the refolding mix as a continuous feed.

3. 3. The method of claim 1 or 2, wherein the refolding mix is ​​incubated for at least 1 hour prior to addition of the heme cofactor.

4. The method according to any one of claims 1 to 3, wherein the heme peroxidase is a class II or class III heme peroxidase.

5. The method according to any one of claims 1 to 4, wherein the heme peroxidase consists of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3 and has heme peroxidase activity.

6. The method of any one of claims 1 to 5, wherein the heme cofactor is hemin.

7. The IB form of heme peroxidase is: - culturing a host cell expressing the gene encoding said heme peroxidase; and - obtaining IBs from said host cells, The method according to any one of claims 1 to 6, provided by

8. The method of any one of claims 1 to 7, wherein the solubilization comprises incubating the IBs in a solubilization buffer.

9. 9. The method of claim 8, wherein the solubilization buffer has a reducing condition corresponding to a dithiothreitol (DTT) concentration of 1 to 50 mmol / L.

10. 10. The method of any one of claims 1 to 9, further comprising the step of purifying the refolded heme peroxidase after the heme cofactor is added to the refolding mix.

11. A method for producing a heme peroxidase product, comprising producing heme peroxidase by the method according to any one of claims 1 to 10.

12. The method of claim 11 further comprising a conjugation reaction to obtain a heme peroxidase conjugate.

13. The method according to claim 11 or 12, further comprising the step of purifying, freezing and / or lyophilizing the heme peroxidase or the heme peroxidase conjugate obtained by the conjugation reaction of the heme peroxidase.

14. The method according to any one of claims 11 to 13, further comprising a step of immobilizing the heme peroxidase or a heme peroxidase conjugate obtained by a conjugation reaction of the heme peroxidase on a solid support.

15. The method according to any one of claims 11 to 14, further comprising packaging the heme peroxidase or a heme peroxidase conjugate obtained by the conjugation reaction of the heme peroxidase into a kit.

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