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

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

Tandem Cell-Free Protein Synthesis as a Tool for Rapid Screening of Optimal Molecular Chaperones.

A simple and flexible method is developed for rapid screening of molecular chaperones that enhance the functional expression of recombinant proteins. A panel of molecular chaperones are transiently expressed in a reaction mixture of cell-free protein synthesis and then a target protein is subsequently expressed in the presence of these presynthesized molecular chaperones. The biological activity of the cell-free synthesized target protein is compared to identify the effective molecular chaperones. This strategy successfully identifies individual and combinations of bacterial molecular chaperones that markedly improved the functional expression of horseradish peroxidase. The authors believe that the presented strategy provides a versatile platform for the optimal production of functional proteins, and can also be extended to studies of other interacting proteins.

Quintuple labeling in the electron microscope with genetically encoded enhanced horseradish peroxidase.

Genetic encoded multilabeling is essential for modern cell biology. In fluorescence microscopy this need has been satisfied by the development of numerous color-variants of the green fluorescent protein. In electron microscopy, however, true genetic encoded multilabeling is currently not possible. Here, we introduce combinatorial cell organelle type-specific labeling as a strategy for multilabeling. First, we created a reliable and high sensitive label by evolving the catalytic activity of horseradish peroxidase (HRP). We then built fusion proteins that targeted our new enhanced HRP (eHRP) to three cell organelles whose labeling pattern did not overlap with each other. The labeling of the endoplasmic reticulum, synaptic vesicles and the plasma membrane consequently allowed for triple labeling in the EM. The combinatorial expression of the three organelle-specific constructs increased the number of clearly distinguishable labels to seven. This strategy of multilabeling for EM closes a significant gap in our tool set and has a broad application range in cell biology.

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris.

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins.

Ultra-high-throughput screening of an in vitro-synthesized horseradish peroxidase displayed on microbeads using cell sorter.

The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases.

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

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

Recombinant horseradish peroxidase: production and analytical applications.

Horseradish peroxidase is a key enzyme in bio- and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli cells and the subsequent refolding of the resulting protein yield a recombinant enzyme that is comparable in its spectral and catalytic characteristics to the native plant peroxidase. Genetic engineering approaches allow production of recombinant peroxidase conjugates with both protein antigens and Fab antibody fragments. The present article reviews the use of recombinant horseradish peroxidase as the marker enzyme in ELISA procedures as well as in amperometric sensors based on direct electron transfer.

Techno-economic analysis of horseradish peroxidase production using a transient expression system in Nicotiana benthamiana.

Despite the advantages of plant-based transient expression systems relative to microbial or mammalian cell systems, the commercial production of recombinant proteins using plants has not yet been achieved to any significant extent. One of the challenges has been the lack of published data on the costs of manufacture for products other than biopharmaceuticals. In this study, we report on the techno-economic analysis of the production of a standard commercial enzyme, namely, horseradish peroxidase (HRP), using a transient expression system in Nicotiana benthamiana. Based on the proven plant yield of 240 mg HRP/kg biomass, a biomass productivity of 15-kg biomass/m(2)/year and a process yield of 54 % (mg HRP product/mg HRP in biomass), it is apparent that HRP can be manufactured economically via transient expression in plants in a large-scale facility (>5 kg HRP/year). At this level, the process is competitive versus the existing technology (extraction of the enzyme from horseradish), and the product is of comparable or improved activity, containing only the preferred isoenzyme C. Production scale, protein yield and biomass productivity are found to be the most important determinants of overall viability.

Heterologous production of horseradish peroxidase C1a by the basidiomycete yeast Cryptococcus sp. S-2 using codon and signal optimizations.

In the present study, we attempted to improve the production of recombinant horseradish peroxidase C1a (HRP-C1a; a heme-binding protein) by Cryptococcus sp. S-2. Both native and codon-optimized HRP-C1a genes were expressed under the control of a high-level expression promoter. When the HRP-C1a gene with native codons was expressed, poly(A) tails tended to be added within the coding region, producing truncated messenger RNAs (mRNAs) that lacked the 3' ends. Codon optimization prevented polyadenylation within the coding region and increased both the mRNA and protein levels of active HRP-C1a. To improve secretion of the recombinant protein, we tested five types of N-terminal signal peptide (NTP). These included the native HRP-C1a NTP (C1a-NTP), short and long xylanase secretion signals (X1-NTP and X2-NTP), cutinase signal (C-NTP), and amylase signal (A-NTP), with and without a C-terminal propeptide (CTP). X2-NTP without CTP resulted in the highest HRP-C1a secretion into the culture medium. HRP-C1a secretion was further increased by using xylose fed-batch fermentation. The production of HRP-C1a in this study was 2.7 and 15 times higher than the production reported in previous studies that used insect cell and Pichia expression systems, respectively.

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

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

Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imaging.

Correlated light and electron microscopic (CLEM) imaging is a powerful method for dissecting cell and tissue function at high resolution. Each imaging mode provides unique information, and the combination of the two can contribute to a better understanding of the spatiotemporal patterns of protein expression, trafficking, and function. Critical to these methods is the use of genetically appended tags that highlight specific proteins of interest in order to be able to pick them out of their complex cellular environment. Here we review and discuss the current generation of genetic labels for direct protein identification by CLEM, addressing their relative strengths and weaknesses and in what experiments they would be most useful.

A dynamic fed batch strategy for a Pichia pastoris mixed feed system to increase process understanding.

Mixed substrate feeding strategies are frequently investigated to enhance the productivity of recombinant Pichia pastoris processes. For this purpose, numerous fed batch experiments or time-consuming continuous cultivations are required to optimize control parameters such as the substrate mixing ratio and the applied methanol concentration. In this study, we decoupled the feeding of methanol and glycerol in a mixed substrate fed batch environment to gain process understanding for a recombinant P. pastoris Muts strain producing the model enzyme horseradish peroxidase. Specific substrate uptake rates (qs) were controlled separately, and a stepwise increased qGly-control scheme was applied to investigate the effect of various substrate fluxes on the culture. The qs-controlled strategy allowed a parallel characterization of the metabolism and the recombinant protein expression in a fed batch environment. A critical-specific glycerol uptake rate was determined, where a decline of the specific productivity occurred, and a time-dependent acceleration of protein expression was characterized with the dynamic fed batch approach. Based on the observations on recombinant protein expression, propositions for an optimal feeding design to target maximal productivities were stated. Thus, the dynamic fed batch strategy was found to be a valuable tool for both process understanding and optimization of product formation for P. pastoris in a mixed substrate environment.

Tumor-targeted gene therapy using Adv-AFP-HRPC/IAA prodrug system suppresses growth of hepatoma xenografted in mice.

Clinical efficacy of current therapies for hepatocellular carcinoma (HCC) treatment is limited. Indole-3-acetic acid (IAA) is non-toxic for mammalian cells. Oxidative decarboxylation of IAA by horseradish peroxidase (HRP) leads to toxic effects of IAA. The purpose of this study was to investigate the effects of a novel gene-targeted enzyme prodrug therapy with IAA on hepatoma growth in vitro and in vivo mouse hepatoma models. We generated a plasmid using adenovirus to express HRP isoenzyme C (HRPC) with the HCC marker, alpha-fetoprotein (AFP), as the promoter (pAdv-AFP-HRPC). Hepatocellular cells were infected with pAdv-AFP-HRPC and treated with IAA. Cell death was detected using MTT assay. Hepatoma xenografts were developed in mice by injection of mouse hepatoma cells. The size and weight of tumors and organs were evaluated. Cell death in tumors was assessed using hematoxylin and eosin-stained tissue sections. HRPC expression in tissues was detected using Reverse Transcriptase-Polymerase Chain Reaction. IAA stimulated death of hepatocellular cells infected with pAdv-AFP-HRPC, in a dose- and time-dependent manner, but not in control cells. Growth of hepatoma xenografts, including the size and weight, was inhibited in mice treated with pAdv-AFP-HRPC and IAA, compared with that in control group. pAdv-AFP-HRPC/IAA treatment induced cell death in hepatoma xenografts in mice. HRPC gene expressed only in hepatoma, but not in other normal organs of mice. pAdv-AFP-HRPC/IAA treatment did not cause any side effects on normal organs. These findings suggest that pAdv-AFP-HRPC/IAA enzyme/prodrug system may serve as a strategy for HCC therapy.

A dynamic method based on the specific substrate uptake rate to set up a feeding strategy for Pichia pastoris.

BACKGROUND: Pichia pastoris is one of the most important host organisms for the recombinant production of proteins in industrial biotechnology. To date, strain specific parameters, which are needed to set up feeding profiles for fed batch cultivations, are determined by time-consuming continuous cultures or consecutive fed batch cultivations, operated at different parameter sets. RESULTS: Here, we developed a novel approach based on fast and easy to do batch cultivations with methanol pulses enabling a more rapid determination of the strain specific parameters specific substrate uptake rate qs, specific productivity qp and the adaption time (Δtimeadapt) of the culture to methanol. Based on qs, an innovative feeding strategy to increase the productivity of a recombinant Pichia pastoris strain was developed. Higher specific substrate uptake rates resulted in increased specific productivity, which also showed a time dependent trajectory. A dynamic feeding strategy, where the setpoints for qs were increased stepwise until a qs max of 2.0 mmol·g-1·h-1 resulted in the highest specific productivity of 11 U·g-1·h-1. CONCLUSIONS: Our strategy describes a novel and fast approach to determine strain specific parameters of a recombinant Pichia pastoris strain to set up feeding profiles solely based on the specific substrate uptake rate. This approach is generic and will allow application to other products and other hosts.

Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase.

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27 °C instead of at 24 °C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots.

High-efficiency secretory production of peroxidase C1a using vesicular transport engineering in transgenic tobacco.

Horseradish peroxidase isozyme C1a (HRP C1a) is widely used as a reporter enzyme in a variety of detection procedures such as enzyme-linked immunosorbent assay (ELISA) and western blotting. We previously isolated the gene encoding HRP C1a and showed that HRP C1a is at first translated as a preproprotein containing propeptides at its N- and C-termini (N-terminal secretion signal peptide and C-terminal propeptide; CTPP). The signal peptide (sp) is necessary for endoplasmic reticulum (ER) translocation and the CTPP acts as a vacuolar sorting determinant. Furthermore, HRP C1a was secreted into the culture medium from cells expressing the HRP C1a gene without the CTPP region. We optimized the secretory production system of HRP C1a in tobacco plants. To determine a suitable signal peptide for high-efficient secretion of proteins, three types of sp derived from HRP C1a (C1Psp), beta-D-glucan exohydrolase (GEsp) and 38 kDa peroxidase (38Psp) were compared. GE and 38P are secretory proteins highly accumulated in the culture medium of BY2 cells. The secretion efficiency was increased by 34% and 53% when GEsp and 38Psp was used, respectively. Next, we used a translational enhancer, the 5'-untranslated region of Nicotiana tabacum alcohol dehydrogenase gene (NtADH 5'-UTR). The production of HRP C1a was increased by placing NtADH 5'UTR in front of the ORF in BY2 cells. These results indicate that the localization and expression level of recombinant proteins can be controlled by the use of propeptides and 5'UTR, respectively. Finally, high-efficiency secretory production of the HRP C1a was also achieved in transgenic tobacco.

Transgenic tobacco (Nicotiana tabacum L. cv. Samsun-NN) plants over-expressing a synthetic HRP-C gene are altered in growth, development and susceptibility to abiotic stress.

The physiological role of class III peroxidases (EC 1.11.1.7) in controlling plant growth and development has been investigated by over-expression of both native and heterologous peroxidases. However, it has remained an enigma as to why the phenotypes of different peroxidase over-expressing transgenics vary. In order to resolve the conflicting information about the consequences of peroxidase over-expression, we have explored the role of the subcellular targeting of HRP-C in controlling stem growth, root development, axillary branching and abiotic stress tolerance in tobacco (Nicotiana tabacum L.). Altering the sub-cellular targeting of vacuolar HRP-C, such that over-expressed peroxidase accumulates in the cytoplasm and cell wall, induced phenotypic changes that are typically associated with altered auxin homeostasis, and over-expression of cell wall located peroxidases. We conclude that sub-cellular targeting is a determinant of the phenotype of peroxidase over-expressing plants.

Large-scale production of hairy root.

Many products of interest are synthesized in organized tissues, but not formed in suspension or callus culture. Therefore, most attention has been focused on root cultures. The transgenic plant,"hairy root", has brought us to dramatic improvements in growth rate and high content of desirable products. Since the roots are quite different from callus in morphology, the culture manner should be explored independently. By providing a growth environment, an elite hairy root can be a more attractive plant. Both of strain selection to generate more competent plants in breeding and engineering development are necessary to overcome various limitations. In this chapter the engineering issues involved in using hairy root culture are discussed, as follows. 1. Measurement of cell concentration on line, and a designing bioreactors for hairy root in liquid culture. 2. High cell density culture and its kinetic parameters. 3. Secretion of target products. 4. The micropropagation of the regenerated hairy root by means of artificial seed system. In some cases where callus and suspension culture show negligible productivity, organ culture will be necessary to achieve good formation. This study on hairy root culture indicates one of the best attempts to the recovery of products from the organ culture in plant biotechnology.

cis-regulatory elements of the peroxidase gene in Arabidopsis thaliana involved in root-specific expression and responsiveness to high-salt stress.

The pattern of organ-specific expression of Arabidopsis thaliana peroxidase was similar to that in horseradish. Tobacco plants transformed with the gus gene fused to the -580 bp deletion (Ea-580) of A. thaliana exhibited high GUS expression in roots. Gel retardation and footprinting analyses showed that at least three domains of fragment between -172 and -1 bp have cis-acting element activities. Several physiological functions for plant peroxidases have been suggested; for example, a metabolic adaptation to salinity in the environment can be induced by certain specific elements of the peroxidase gene. The prxEa promoter fragments (Ea-580 and Ea-390) show multiple cis-acting elements in the control of expression in high-salt stress. These data suggest that the DNA-binding factor may be involved in the regulation of gene expression in specific organ and salt stress.

Construction of gene expression system in cultured tobacco cells.

To construct a gene expression system in cultured tobacco cells, useful regulatory elements of plant genes were studied. The promoter of the horseradish peroxidase gene, prxC2, showed high activity in tobacco cells, and it contained enhancer sequences and a cis element for wound induction. The heat shock promoter of the HSP18.2 gene from A. thaliana had strong activity of transcription when the incubation temperature of tobacco cells was shifted from 25 degrees C to 37 degrees C. These elements could be good candidates for foreign gene expression in tobacco cells.