Production of recombinant allergens in plants

A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed.     

Over-expression and production of plant allergens by molecular farming strategies

Recombinant allergens have become a valuable tool for diagnosis and may also be used for therapy in the near future. To supply the required large amounts of functional recombinant proteins on a cost-effective basis, the production of allergens in plants by molecular farming is an alternative to microbial expression systems. Especially as post-translational modifications of the allergens, e.g., phosphorylation and glycosylation, may be important for recognition by the human immune system, the plant-based production of recombinant allergens enables the correct folding, glycosylation, and other modifications of the recombinant allergen. An introduction to the methods for plant transformation via the tumor-inducing bacterium, Agrobacterium tumefaciens, is given in this paper.     

Inteins and affinity resin substitutes for protein purification and scale up

The development of self-cleaving fusion-tag technology has greatly simplified the purification of recombinant proteins at laboratory scale. The self-cleaving capability of these tags has recently been combined with additional purification tags to generate novel and convenient protein purification methods at a variety of scales. In this review, we describe some of these methods, and provide a rudimentary economic analysis of hypothetical large-scale applications. This work is expected to provide a rough outline for the evaluation of these methods for large-scale bioprocessing of a variety of products.     

Oleosin fusion expression systems for the production of recombinant proteins

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals at large scale.     

Quantitation of latex allergens

Minimizing allergen concentration in latex goods to prevent sensitization to natural rubber latex (NRL) and thereby the development of clinical allergy is acknowledged as of mutual interest for rubber manufacturers and regulatory health authorities. However, measuring total protein, the principal currently available method, cannot be deemed a satisfactory regulatory measure to control allergen content. Specific methods based on human IgE-containing reagents, such as radioallergosorbent test (RAST) inhibition, have been available in certain laboratories for demonstrating NRL allergens in rubber products but the methods lack standardization. Currently, one commercial test has become available for measuring individual NRL allergens by capture ELISA-based assays using monoclonal antibodies and purified or recombinant allergens. Such methods are specific, they can be properly standardized, and they are of sufficient sensitivity and reproducibility. Results from medical gloves collected in two national market surveys in Finland in 1995 and 1999, respectively, show that Hev b 6.02 and Hev b 5, the two major allergens for NRL-allergic adults, are the most abundant allergens regularly detectable in high- and moderate-allergen gloves. In addition, Hev b 3 and Hev b 1, the two major allergens for children with spina bifida, are also commonly found. In general, when the sum of the four allergens exceeded 1 microg/g, most NRL-allergic patients showed positive skin prick test reactions against them. Using these new methods assessment of threshold levels that could in due course become guidelines for the rubber industry and regulatory health authorities is becoming possible. Eventually, this progress is expected to lead to a declining incidence of latex allergy.     

Provocation testing with recombinant allergens

In the past few decades, DNA technology has enabled the production of defined recombinant allergen molecules for diagnostic and therapeutic purposes. Recombinant allergens containing most of the relevant IgE epitopes present in natural allergen sources are now available and allergen proteins can be produced that are identical, without biological or batch-to-batch variation. A great advantage of recombinant allergens is that they can be used for component-resolved diagnostics, which makes it possible to establish the patient's individual IgE reactivity profile before therapy is selected. However, before recombinant allergens can be applied in clinical practice their biological activity has to be carefully investigated in vivo. We here describe the most commonly used provocation methods (skin tests (prick and intradermal), nasal, bronchial, and conjunctival provocations) and how they can be performed. We also discuss the results so far obtained with in vivo testing using recombinant allergens and envisage their future use for immunotherapy.     

Recovery and purification of plant-made recombinant proteins

Plants are becoming commercially acceptable for recombinant protein production for human therapeutics, vaccine antigens, industrial enzymes, and nutraceuticals. Recently, significant advances in expression, protein glycosylation, and gene-to-product development time have been achieved. Safety and regulatory concerns for open-field production systems have also been addressed by using contained systems to grow transgenic plants. However, using contained systems eliminates several advantages of open-field production, such as inexpensive upstream production and scale-up costs. Upstream technological achievements have not been matched by downstream processing advancements. In the past 10 years, the most research progress was achieved in the areas of extraction and pretreatment. Extraction conditions have been optimized for numerous proteins on a case-by-case basis leading to the development of platform-dependent approaches. Pretreatment advances were made after realizing that plant extracts and homogenates have unique compositions that require distinct conditioning prior to purification. However, scientists have relied on purification methods developed for other protein production hosts with modest investments in developing novel plant purification tools. Recently, non-chromatographic purification methods, such as aqueous two-phase partitioning and membrane filtration, have been evaluated as low-cost purification alternatives to packed-bed adsorption. This paper reviews seed, leafy, and bioreactor-based platforms, highlights strategies for the primary recovery and purification of recombinant proteins, and compares process economics between systems. Lastly, the future direction and research needs for developing economically competitive recombinant proteins with commercial potential are discussed.     

A comprehensive system for protein purification and biochemical analysis based on antibodies to c-myc peptide.

The genomics revolution has created a need for increased speed and generality for recombinant protein production systems as well as general methods for conducting biochemical assays with the purified protein products. 9E10 is a well-known high-affinity antibody that has found use in a wide variety of biochemical assays. Here we present a standardized system for purifying proteins with a simple epitope tag based on c-myc peptide using an antibody affinity column. Antibodies with binding parameters suitable for protein purification have been generated and characterized. To purify these antibodies from serum-containing medium without carrying through contaminating immunoglobulin G, a peptide-based purification process was developed. A fluorescence polarization binding assay was developed to characterize the antigen-antibody interaction. Protein purification protocols were optimized using a fluorescein-labeled peptide as a surrogate "protein." Binding and elution parameters were evaluated and optimized and basic operating conditions were defined. Several examples using this procedure for the purification of recombinant proteins are presented demonstrating the generality of the system. In all cases tested, highly pure final products are obtained in good yields. The combination of the antibodies described here and 9E10 allow for almost any biochemical application to be utilized with a single simple peptide tag.     

Production of Recombinant Peanut Allergen Ara h 2 using <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis.     

Recombinant allergens for analysing T-cell responses

T-cell responses constitute a central element of allergic disease and a model for studying Th1 and Th2 cytokine pathways. Most studies to date have used extracts of allergens which contain variable quantities of different allergens and non-allergenic antigens. Recombinant allergens provide the tools for studying the responses to allergens in a reproducible and dose-dependent manner and the different T-cell responses of allergic and non-allergic subjects provide a method for verifying the responses and their relationship to allergic sensitisation. Most allergies show dominant responses to one or a few major allergens. These allergens have been described for the common allergies and have been produced as recombinant allergens. A particular problem for allergens is that many are mixtures of proteins from multi-gene families or are highly polymorphic. Information now exists so the sequence variation can be represented. Purified recombinant allergens produced by standard expression systems stimulate the expected T-cell responses from the peripheral blood of allergic and non-allergics to allergen extracts. Although stimulation with recombinant allergens which are not produced with a natural IgE binding activity can provide a measure of allergenicity, the altered tertiary structure can reduce Th2 responses. The sequence information now available provides the means to use PCR to produce cDNA for the production of recombinant allergens from readily available sources. The production of the highly reactive recombinant Der p 2 allergen of house dust mite from natural sources is described.     

Transition of recombinant allergens from bench to clinical application

The cloning and production of an increasing number of allergens through the use of DNA technology has provided the opportunity to use these proteins instead of natural allergen extracts for the diagnosis and therapy of IgE-mediated allergic disease. For diagnostic purposes, it is essential that the molecules exhibit IgE-reactivity comparable with that of the natural wild-type molecules, whereas T cell reactivity and immunogenic activity may be more important for allergen-specific immunotherapy. In relation to the latter, the development of hypoallergenic recombinant allergen variants is an approach which shows great promise. Clinical application of the proteins requires that they must be produced under conditions of Good Manufacturing Practice and meet the specifications set down in the appropriate Regulatory Guidelines, principally the ICH-Guidelines. Special consideration has to be given to the choice of expression system, the design of the expression vectors, and the purification strategy to obtain a pure product free from toxins and contamination. The availability of the pure recombinant molecules provides the opportunity to formulate preparations that are free from the non-allergenic ballast proteins present in natural allergen extracts and which contain relative concentrations of the allergens in clinically appropriate proportions.     

Characterization of recombinant human cathepsin B expressed at high levels in baculovirus.

The lysosomal cysteine protease cathepsin B has been studied intensely for many years because of its unique characteristics and its potential involvement in disease states. A reproducible, high yield expression system for active recombinant protein is key to biochemical and biophysical studies as well as rational drug design. Although several microbial and mammalian expression systems for recombinant human cathepsin B have been described, these have been limited by low or variable yields. Further, in some of these systems hyper-glycosylation of the enzyme near the active site affects its activity. We describe a baculovirus expression system and purification scheme that solve all of these problems. Yields of active, protected enzyme were reproducibly in excess of 25 mg/L. Since this protein was not hyper-glycosylated, it had greater activity than cathepsin B produced in yeast systems as indicated by a threefold increase in Kcat. In addition, the biophysical properties of the baculovirus-expressed cathepsin B, as measured by dynamic light scattering, were more amenable to crystallographic study since the data indicated proteins of more uniform size. Therefore, this system for the production of recombinant human cathepsin B constitutes a major improvement in both quantity and quality over those previously reported. Further, we demonstrate that the manner of expression and purification of this enzyme has profound effects on its kinetic and physical parameters.     

Heterologous expression of sarcoplasmic reticulum Ca2+-ATPase

In recent years, expression of rabbit sarcoplasmic reticulum (SR) Ca²⁺-ATPase in heterologous systems has been a widely used strategy to study altered enzymes generated by site-directed mutagenesis. Various eukaryotic expression systems have been tested, all of them yielding comparable amounts of recombinant protein. However, the relatively low yield of recombinant protein obtained so far suggests that novel purification techniques will be required to allow further characterization of this enzyme based on direct ligand-binding measurements.     

Rapid purification of bacterially expressed fusion proteins by high-performance liquid chromatography methods

Recent developments in recombinant DNA technology allow the high-level expression in bacteria of substantial amounts of viral and eukaryotic proteins whose genes have been cloned into plasmids. The present study reports two high-performance liquid chromatography (HPLC) methods for the rapid purification to apparent homogeneity of these bacterially expressed proteins. The two methods are anion exchange HPLC in the presence of 7 M Urea and reverse-phase HPLC of protein solubilized by 7.0 M guanidine hydrochloride. The two methods have been used successfully to purify fusion products of the v-myb oncogene and fusion proteins from HTLV-I Px and transmembrane regions and should be of general utility for purification of other bacterially produced proteins.     

Microarrayed allergens for IgE profiling

Diagnosis of type I allergy is based on anamnesis, provocation testing, and serological determination of total and specific IgE. Currently, in vivo and in vitro diagnostic tests employ allergen extracts prepared from various allergen sources (e.g., pollen, mites, animal dander, moulds, foods, venoms, etc.). The application of recombinant DNA technology to the field of allergen characterization has allowed to reveal the molecular nature of the most common allergens. To date a continuously increasing number of allergen sequences has become available and panels of recombinant allergens-assembling the epitope complexity of natural allergens sources-can be produced. The use of recombinant allergens instead of crude, natural extracts for allergy diagnosis allows us to determine the individual IgE reactivity profile of each patient. To enable a comprehensive analysis of the patient's IgE binding pattern to a large number of individual allergens, a new type of serological test is required. In this paper, we applied microarray technology to create a multi-allergen test system, based on microarrayed recombinant allergens.     

Expression and purification of rotavirus proteins NSP5 and NSP6 in Escherichia coli

Rotaviruses are one of the worldwide leading causes of gastroenteritis in children under 5 yr old. The rotavirus nonstructural NSP5 is a phosphoprotein implicated in viroplasms formation, whereas NSP6 could have a possible regulatory role of NSP5. It has been reported that N- and C-termini of NSP5 are important for amount of protein is required for structural analysis, efficient expression systems are required. His-tag fusion at the C-terminus and glutathione-S-transferase (GST)-fusion at the N-terminus were used as expression systems, and conditions for recombinant proteins expression were obtained. His-tag fusion was not efficient to produce NSP5 (2% of total protein), but NSP6 was expressed in higher amounts (11% of total protein). In contrast, GST-NSP5 and GST-NSP6 proteins correspond to 34 and 31% of the total proteins, respectively. GST-fusions seem to have a protective effect against nonstructural rotavirus protein toxicity in Escherichia coli; however, in both systems, NSP5 and NSP6 recombinant proteins were expressed as inclusion bodies. Conditions for solubilization and purification of recombinant proteins were achieved. This is the first report of expression and purification of NSP5 and NSP6 recombinant proteins in suitable amounts for further structural analysis.     

A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins

We have developed and evaluated a highly parallel protein expression and purification system using ORFs derived from the pathogenic bacterium Streptococcus pneumoniae as a representative test case in conjunction with the Gateway cloning technology. Establishing high throughput protein production capability is essential for genome-wide characterization of protein function. In this study, we focused on protein expression and purification outcomes generated from an expression vector which encodes an NH(2)-terminal hexa-histidine tag and a COOH-terminal S-tag. Purified recombinant proteins were validated by SDS-PAGE, followed by in-gel digestion and identification by MALDI-TOF/TOF analysis. Starting with 1360 sequence-validated destination clones we examined correlation analyses of expression and solubility of a wide variety of recombinant proteins. In total, 428 purified proteins (31%) were recovered in soluble form. We describe a semi-quantitative scoring method using an S-tag assay to improve the throughput and efficiency of expression and solubility studies for recombinant proteins. Given a relatively large dataset derived from proteins representing all functional groups in a microbial genome we correlated various protein characteristics as they relate to protein expression outcomes.     

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Manufacturing practices for recombinant adeno‐associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10⁴–10⁵ vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small‐scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol‐step gradient whereas large‐scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead‐end filtration) and chromatography based‐methods are used for large‐scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.