Valuable Recombinant Proteins Research Articles

Chromatography affinity resin with photosynthetically-sourced protein A ligand

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.

Current state of research in the development of the genomic editing method: problems and prospects

The possibility of using the CRISPR/Cas method of genomic editing has provided researchers with a powerful tool not only for targeted modification of genes that determine economically valuable traits in plants, but also for solving fundamental problems of their functioning. The most striking examples of the use of CRISPR/Cas9 to improve various plant species by knockouts of target genes or knockins of expression cassettes, including genes that change the biosynthesis of important plant metabolites, obtained by foreign research groups, are presented. We discuss our own results on the directed change in the functioning of genes encoding photosystem II carbonic anhydrases, as well as genes involved in plant responses to stress in theArabidopsis thalianamodel. Examples of the use of the genomic editing method to improve the characteristics of plant cell cultures as bioproducers of pharmaceutically valuable recombinant proteins are given. Methodological issues related to plant genome editing are considered — the problems of chimerism, obtaining homozygotes and biallelic knockout mutations, knockout of regulatory and structural genes, as well as repair features in the regions of integration of expression cassettes in knockins. The main directions for further development and improvement of the CRISPR/Cas genomic editing method aimed at optimizing the efficiency of delivery of target genetic constructs and editing tools to the nuclear and chloroplast genomes of plants using single-walled carbon nanotubes are summarized.

Applications of CRISPR/Cas9 in a rice protein expression system via an intron-targeted insertion approach

The sugar starvation-inducible rice αAmy3 promoter and signal peptide are widely used to produce valuable recombinant proteins in rice suspension culture cells. Conventionally, the recombinant gene expression cassette is inserted into the genome at random locations by Agrobacterium- or particle bombardment-mediated transformation. CRISPR/Cas9 gene editing enables gene insertion at a precise target site in the genome. In this study the CRISPR/Cas9 approach was modified for intron-targeted insertion by adding an artificial 3′ splicing site upstream of the recombinant gene. Knock-in transgenic rice cell lines containing the recombinant GFP gene inserted in intron 1 of αAmy3 were generated. The endogenous αAmy3 promoter regulated recombinant gene expression and the αAmy3 signal peptide directed secretion of the recombinant GFP protein into the culture medium. In addition, the recombinant GFP protein was localized in amyloplasts, identical to the subcellular localization of endogenous αAmy3 reported previously. This modified CRISPR/Cas9 knock-in approach is simple and highly efficient, and the recombinant gene insertion frequency attained 12.5%. The approach can be applied in the production of pharmaceutical proteins in rice suspension cell cultures. The high efficiency of the GFP reporter gene knock-in method and the maintenance of target gene behavior also make the strategy applicable to endogenous gene functional studies in rice.

Genetically modified animals for use in biopharmacology: from research to production


 
 Introduction: In this review, the analysis of technologies for obtaining biologically active proteins from various sources is carried out, and the comparative analysis of technologies for creating producers of biologically active proteins is presented. Special attention is paid to genetically modified animals as bioreactors for the pharmaceutical industry of a new type. The necessity of improving the technology of development transgenic rabbit producers and creating a platform solution for the production of biological products is substantiated.
 The advantages of using TrB for the production of recombinant proteins: The main advantages of using TrB are the low cost of obtaining valuable complex therapeutic human proteins in readily accessible fluids, their greater safety relative to proteins isolated directly from human blood, and the greater safety of the activity of the native protein.
 The advantages of the mammary gland as a system for the expression of recombinant proteins: The mammary gland is the organ of choice for the expression of valuable recombinant proteins because milk is easy to collect in large volumes.
 Methods for obtaining transgenic animals: The modern understanding of the regulation of gene expression and the discovery of new tools for gene editing can increase the efficiency of creating bioreactors for animals and help to obtain high concentrations of the target protein.
 The advantages of using rabbits as bioreactors producing recombinant proteins in milk: The rabbit is a relatively small animal with a short duration of gestation, puberty and optimal size, capable of producing up to 5 liters of milk per year per female, receiving up to 300 grams of the target protein.
 
 

Exposure of Agrobacterium tumefaciens to agroinfiltration medium demonstrates cellular remodelling and may promote enhanced adaptability for molecular pharming.

Agroinfiltration is used to treat plants with modified strains of Agrobacterium tumefaciens for the purpose of transient in planta expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently expressed target protein products, as prolonged exposure to agroinfiltration medium suggests remodelling of the bacterial proteins towards enhanced infection of plant cells.

MCherry Protein as an In Vivo Quantitative Reporter of Gene Expression in the Chloroplast of Chlamydomonas reinhardtii

Microalgal chloroplasts have a substantial potential as a sustainable alternative to conventional hosts for recombinant protein production, due to their photosynthetic ability. However, realization of microalgal chloroplast as a platform for the production of recombinant proteins has suffered from difficulties in genetic manipulation and development of molecular tools, including reporter proteins. Here, we investigated the suitability of a fluorescent protein, mCherry, as a reporter for quantitative in vivo monitoring of gene expression in the chloroplast of Chlamydomonas reinhardtii. By analyzing cell growth, the fluorescence intensity of a mCherry-expressing strain, as well as auto-fluorescence, under different photoautotrophic culture conditions, we demonstrated a strong correlation between the fluorescence intensity of mCherry expressed in the chloroplast and its protein expression level. In addition, we found that the supply of CO2 and light energy can be an important factor for the synthesis of recombinant proteins in the microalgal chloroplast. Our results identified mCherry as a reliable and quantitative reporter for the study of gene expression in chloroplasts, which is essential for the biotechnological application of microalgal chloroplasts and for improved production of valuable recombinant proteins.

Recovery of active recombinant EGFP from the excrement of transgenic mice: A possible source of recombinant protein

Transgenic animals provide attractive systems for the production of valuable recombinant proteins. Previous studies indicate that milk is a suitable source of secreted recombinant proteins. In the current study, we examine whether excrement can be another source of recombinant proteins by using transgenic mice ubiquitously-expressing green fluorescent protein (GFP) as a model. We found that the surface of excrement from GFP-transgenic mice was fluorescent, and the supernatant after centrifugation of an excrement suspension was rich in undegraded, actively fluorescing GFP. GFP was successfully purified from stool as a fluorescent 27 kDa protein by using a common procedure. Finally, we observed that the fluorescence of digested materials began in the ileum and persisted throughout the remainder of the digestive tract. Our results demonstrate that excrement, which is produced daily regardless of the sex or age of the animal, may be another feasible source of recombinant proteins. The preparation method is simple, economical, and noninvasive.

Transient expression of a bovine leukemia virus envelope glycoprotein in plants by a recombinant TBSV vector

Plants offer a unique combination of advantages for the production of valuable recombinant proteins in a relatively short time. For instance, a variety of diagnostic tests have been developed that use recombinant antigens expressed in plants. The envelope glycoprotein gp51 encoded by Bovine leukemia virus (BLV) is one of the essential subunits for viral infectivity. It was indicated that the recombinant gp51 (rgp51) of BLV сan be used as an synthetic alternative antigen useful in the diagnosis of BLV infection in cattle. Here we evaluate the potential for using a viral vector based on the genome of Tomato bushy stunt virus (TBSV) for the efficient expression of BLV envelope glycoprotein rgp51 in Nicotiana benthamiana plants. The codon-optimized gene encoding rgp51 was synthesized by the de novo DNA synthesis to replace the GFP gene in the TBSV-derived viral vector that was then delivered into 4–5 week old N. benthamiana plants by agroinfiltration. Expression of recombinant his-tagged rgp51 was verified by protein extraction followed by western blot procedures, and by purification using Ni2+-affinity chromatography. The molecular weight of this plant-expressed rgp51 ranged from 43 to 55 kDa and it was shown to be glycosylated. Important for potential use in diagnostic tests, purified rgp51 specifically reacted with BLV infected bovine sera while no reaction was observed with the negative serum samples.

Expression of camel (Camelus bactrianus) and bovine (Bos Taurus) chymosin in Pichia (Komagataella) pastoris

Plants offer a unique combination of advantages for the production of valuable recombinant proteins in a relatively short time. For instance, a variety of diagnostic tests have been developed that use recombinant antigens expressed in plants. The envelope glycoprotein gp51 encoded by Bovine leukemia virus (BLV) is one of the essential subunits for viral infectivity. It was indicated that the recombinant gp51 (rgp51) of BLV сan be used as an synthetic alternative antigen useful in the diagnosis of BLV infection in cattle. Here we evaluate the potential for using a viral vector based on the genome of Tomato bushy stunt virus (TBSV) for the efficient expression of BLV envelope glycoprotein rgp51 in Nicotiana benthamiana plants. The codon-optimized gene encoding rgp51 was synthesized by the de novo DNA synthesis to replace the GFP gene in the TBSV-derived viral vector that was then delivered into 4–5 week old N. benthamiana plants by agroinfiltration. Expression of recombinant his-tagged rgp51 was verified by protein extraction followed by western blot procedures, and by purification using Ni2+-affinity chromatography. The molecular weight of this plant-expressed rgp51 ranged from 43 to 55 kDa and it was shown to be glycosylated. Important for potential use in diagnostic tests, purified rgp51 specifically reacted with BLV infected bovine sera while no reaction was observed with the negative serum samples.

Seed specific expression and analysis of recombinant human adenosine deaminase (hADA) in three host plant species.

The plant seed is a leading platform amongst plant-based storage systems for the production of recombinant proteins. In this study, we compared the activity of human adenosine deaminase (hADA) expressed in transgenic seeds of three different plant species: pea (Pisum sativum L.), Nicotiana benthamiana L. and tarwi (Lupinus mutabilis Sweet). All three species were transformed with the same expression vector containing the hADA gene driven by the seed-specific promoter LegA2 with an apoplast targeting pinII signal peptide. During the study, several independent transgenic lines were generated and screened from each plant species and only lines with a single copy of the gene of interest were used for hADA expression analysis. A stable transgenic canola line expressing the ADA protein, under the control of 35S constitutive promoter was used as both as a positive control and for comparative study with the seed specific promoter. Significant differences were detected in the expression of hADA. The highest activity of the hADA enzyme (Units/g seed) was reported in tarwi (4.26U/g) followed by pea (3.23U/g) and Nicotiana benthamiana (1.69U/g). The expression of mouse ADA in canola was very low in both seed and leaf tissue compared to other host plants, confirming higher activity of seed specific promoter. Altogether, these results suggest that tarwi could be an excellent candidate for the production of valuable recombinant proteins.

Tackling Unwanted Proteolysis in Plant Production Hosts Used for Molecular Farming.

Although the field of molecular farming has significantly matured over the last years, some obstacles still need to be resolved. A major limiting factor for a broader application of plant hosts for the production of valuable recombinant proteins is the low yield of intact recombinant proteins. These low yields are at least in part due to the action of endogenous plant proteases on the foreign recombinant proteins. This mini review will present the current knowledge of the proteolytic enzymes involved in the degradation of different target proteins and strategies that are applied to suppress undesirable proteolytic activities in order to safeguard recombinant proteins during the production process.

Production of Recombinant Proteins in the Chloroplast of the Green Alga Chlamydomonas reinhardtii.

Chloroplast transformation in the green algae Chlamydomonas reinhardtii can be used for the production of valuable recombinant proteins. Here, we describe chloroplast transformation of C. reinhardtii followed by protein detection. Genes of interest integrate stably by homologous recombination into the chloroplast genome following introduction by particle bombardment. Genes are inherited and expressed in lines recovered after selection in the presence of an antibiotic. Recombinant proteins can be detected by conventional techniques like immunoblotting and purified from liquid cultures.

A Comparison of transgenic and wild type soybean seeds: analysis of transcriptome profiles using RNA-Seq.

BackgroundSoybean (Glycine max) has been bred for thousands of years to produce seeds rich in protein for human and animal consumption, making them an appealing bioreactor for producing valuable recombinant proteins at high levels. However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood. To address this, we investigated whether gene expression within transgenic soybean seed tissue is altered when large amounts of recombinant proteins are being produced and stored exclusively in the seeds. We used RNA-Seq to survey gene expression in three transgenic soybean lines expressing recombinant protein at levels representing up to 1.61 % of total protein in seed tissues. The three lines included: ST77, expressing human thyroglobulin protein (hTG), ST111, expressing human myelin basic protein (hMBP), and 764, expressing a mutant, nontoxic form of a staphylococcal subunit vaccine protein (mSEB). All lines selected for analysis were homozygous and contained a single copy of the transgene.MethodsEach transgenic soybean seed was screened for transgene presence and recombinant protein expression via PCR and western blotting. Whole seed mRNA was extracted and cDNA libraries constructed for Illumina sequencing. Following alignment to the soybean reference genome, differential gene expression analysis was conducted using edgeR and cufflinks. Functional analysis of differentially expressed genes was carried out using the gene ontology analysis tool AgriGO.ResultsThe transcriptomes of nine seeds from each transgenic line were sequenced and compared with wild type seeds. Native soybean gene expression was significantly altered in line 764 (mSEB) with more than 3000 genes being upregulated or downregulated. ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively. Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport. No significant gene ontology terms were detected in ST77, and only a few genes involved in photosynthesis and thylakoid functions were downregulated in ST111. Despite these differences, transgenic plants and seeds appeared phenotypically similar to non-transgenic controls. There was no correlation between recombinant protein expression level and the quantity of differentially expressed genes detected.ConclusionsMeasurable unscripted gene expression changes were detected in the seed transcriptomes of all three transgenic soybean lines analyzed, with line 764 being substantially altered. Differences detected at the transcript level may be due to T-DNA insert locations, random mutations following transformation or direct effects of the recombinant protein itself, or a combination of these. The physiological consequences of such changes remain unknown.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0207-z) contains supplementary material, which is available to authorized users.

Tobacco seeds as efficient production platform for a biologically active anti-HBsAg monoclonal antibody.

The use of plants as heterologous hosts is one of the most promising technologies for manufacturing valuable recombinant proteins. Plant seeds, in particular, constitute ideal production platforms for long-term applications requiring a steady supply of starting material, as they combine the general advantages of plants as bioreactors with the possibility of biomass storage for long periods in a relatively small volume, thus allowing manufacturers to decouple upstream and downstream processing. In the present work we have used transgenic tobacco seeds to produce large amounts of a functionally active mouse monoclonal antibody against the Hepatitis B Virus surface antigen, fused to a KDEL endoplasmic reticulum retrieval motif, under control of regulatory sequences from common bean (Phaseolus vulgaris) seed storage proteins. The antibody accumulated to levels of 6.5mg/g of seed in the T3 generation, and was purified by Protein A affinity chromatography combined with SEC-HPLC. N-glycan analysis indicated that, despite the KDEL signal, the seed-derived plantibody bore both high-mannose and complex-type sugars that indicate partial passage through the Golgi compartment, although its performance in the immunoaffinity purification of HBsAg was unaffected. An analysis discussing the industrial feasibility of replacing the currently used tobacco leaf-derived plantibody with this seed-derived variant is also presented.

Comparison of total soluble protein in various horticultural crops and evaluation of its quantification methods

Various horticultural crops have been considered as potential heterologous expression systems for plant-made recombinant pharmaceutical proteins. However, there is little information concerning the total soluble protein (TSP) levels of major horticultural crops. Ten major horticultural crops-Chinese cabbage, broccoli, garlic, onion, cabbage, bunching onion, cucumber, zucchini, radish, and carrot, along with tobacco and Arabidopsis, were selected to investigate the TSP levels in their individual tissues. In tobacco, SDS-PAGE assay showed that leaf tissues had stronger protein bands than stem tissues, and freshly harvested samples had slightly stronger band density than the −70°C frozen samples, suggesting that fresh leaf should be used to measure the total soluble proteins without any protein loss or degradation. Bicinchoninic acid (BCA) protein assay revealed that among various horticultural crops, garlic (41.4 mg·g−1 FW), broccoli (21.9 mg·g−1 FW), and Chinese cabbage (11.9 mg·g−1 FW) had the highest TSP levels suggesting that these horticultural crops could be good candidates for plant molecular biofarming to produce highly valuable recombinant proteins. The inner clove tissue in garlic, the flower tissue in broccoli, and the green leaf tissue in Chinese cabbage showed the strongest protein band density as compared to other tissues. The TSP of Arabidopsis tissues was quantified by SDS-PAGE, BCA, and Nano-drop methods. In general, the middle leaf tissue showed the highest TSP levels. To evaluate TSP levels of various horticultural crops, these three different methods were compared. The correlation and regression analyses between SDS-PAGE and BCA, and SDS-PAGE and Nano-drop suggested that there were significant correlations between SDS-PAGE and BCA protein assays as compared to SDS-PAGE and Nano-drop assays, indicating that BCA assay is reliable to quantify TSP levels. In conclusion, the TSP levels varied depending on the horticultural crops and their tissue types, and BCA assay could be applied to quantify the TSP.

Molecular Farming – Production of pharmaceuticals in plants and plant cells

Many different plant-based systems have been used to produce valuable small molecules or recombinant proteins but only a small number have made the leap from an experimental platform to a viable commercial process. This reflects a combination of factors, principally the technical issues that must be addressed to achieve competitive performance, the economic principles that need to be satisfied to ensure manufacturing processes are financially viable and sustainable, and the regulatory demands that must be met to ensure that products manufactured in plants are safe, efficacious and meet the quality standards demanded by the regulators [1,2]. In general, plants and plant cells are ideal production platforms, which can be easily scaled up to provide valuable products for pharmaceutical, cosmetic and industrial applications. Typically plant systems are grown in containment under defined conditions, either in the greenhouse or in bioreactors, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. In this presentation, the advantages and disadvantages of commonly used plant-based systems are presented as well as strategies for recovering the final product. In many cases production and downstream processes have to be optimized to establish a commercially viable platform and therefore novel strategies for strain and process optimization, helping to increase yields and scalability are presented. These approaches include the optimization of expression vectors and recombinant protein targeting [3], the testing of different plant platforms [4], the use of statistical experimental techniques to optimise medium and cultivation conditions [5], establishment of elite lines by flow cytometry [6], as well as strategies for scale up and GMP-compliant production [7].

Recombinant biosynthesis of functional human growth hormone and coagulation factor IX in transgenic soybean seeds

Background Plants constitute promising systems for the alternative production of valuable recombinant proteins. Recently, ELELYSOTM, an enzymatic drug developed by the Israeli company Protalix Biotherapeutics to treat Type 1 Gaucher’s disease, became the first plant-derived therapeutic product to reach marketable status [1]. This confirms the inherent potential of plant systems for the large-scale production of pharmaceuticals and industrial proteins with high quality and at competitive costs. Soybeans [Glycine max (L.) Merrill] provide a potential economically viable platform for the large-scale production of different therapeutic molecules. Plant seeds are specialized in the stable accumulation of proteins at high levels, representing an excellent source of abundant and cheap biomass. Additionally, the vegetative growth of plants can be significantly extended under a daily photoperiod of 23 h of light, inducing more than a tenfold increase in seed production when compared with plants cultivated under field conditions [2]. In this report we present the recombinant biosynthesis and the molecular and functional characterization of two important therapeutic proteins in transgenic soybean seeds: the 22 kDa human growth hormone (hGH) and the 56 kDa human coagulation factor IX (hFIX), a vitamin K-dependent serine-protease glycoprotein utilized to treat Type B Christmas disease, the second most frequent haemophilia variant [3,4].

The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori.

The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It's a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed.

When the boundaries between physics and biology blur: A promising future for fungi as producers of valuable recombinant proteins: Reply to comments on: “Physical methods for genetic transformation of fungi and yeast”

We appreciate the commentaries to our review [1], received from Cruz Hernandez et al. [2], Ortiz-Vazquez [3],Arellano-Carbajal [4], Castano [5],Gold[6], and Moosavi-Nejad and Hosseini [7], who express interesting points ofview, enriching the discussion with their expertise.Fungi constitute a kingdom that can be found all around our planet since prehistoric times. Cruz Hernandez andcolleagues [2] comment on interesting historical aspects of fungi in humans’ life and emphasize that nowadays fungiplay a crucial role in basic scientific research, applied sciences, industry and economy. The impressive economicestimate of the total global market for fungal and yeast products, referred to by these authors, supports this. There arestill many areas for improvement in the field of fungal transformation and many groups around the world are activelyworking toward this goal.

Molecular farming of human cytokines and blood products from plants: Challenges in biosynthesis and detection of plant‐produced recombinant proteins

Plants have emerged as an attractive alternative to the traditional mammalian cell cultures or microbial cell-based systems system for the production of valuable recombinant proteins. Through recombinant DNA technology, plants can be engineered to produce large quantities of pharmaceuticals and industrial proteins of high quality at low costs. The recombinant production, by transgenic plants, of therapeutic proteins normally present in human plasma, such as cytokines, coagulation factors, anticoagulants, and immunoglobulins, represents a response to the ongoing challenges in meeting the demand for therapeutic proteins to treat serious inherited or acquired bleeding and immunological diseases. As the clinical utilization of fractionated plasma molecules is limited by high production costs, using recombinant biopharmaceuticals derived from plants represents a feasible alternative to provide efficient treatment. Plant-derived pharmaceuticals also reduce the potential risks to patients of infection with pathogens or unwanted immune responses due to immunogenic antigens. In this review, we summarize the recent advances in molecular farming of cytokines. We also examine the technological basis, upcoming challenges, and perspectives for the biosynthesis and detection of these molecules in different plant production platforms.