Platform For Production Of Recombinant Proteins Research Articles

Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has been shown to hold significant promise as a production platform for recombinant proteins, but transformation of the nuclear genome is still a non-trivial process due to random gene insertion and frequent silencing. Insertion of transgenes into the chloroplasts is an alternative strategy, and we report here the stable expression of a large (91 kDa) protein in the chloroplast using a recently developed low-cost transformation protocol. Moreover, selection of transformants is based on restoration of prototrophy using an endogenous gene (psbH) as the marker, thereby allowing the generation of transgenic lines without the use of antibiotic-resistance genes. Here, we have expressed a bifunctional diterpene synthase in C. reinhardtii chloroplasts. Homoplasmic transformants were obtained with the expressed enzyme accounting for 3.7 % of total soluble protein. The enzyme was purified to homogeneity and expression was shown to have a small but reproducible effect on growth rate at the end of log phase growth. These results demonstrate that large recombinant enzymes can be synthesised in the algal chloroplast, and serve to underline its potential as a platform for the biosynthesis of novel metabolites.

Hydroxyproline-O-glycosylated peptide tags enhance recombinant protein yields in tobacco transient expression

Plant transient expression provides a rapid production platform for recombinant proteins but is linked with low protein yields. To test if plant-specific hydroxyproline (Hyp)-O-glycosylated peptide tags attached to a target protein can improve overall yields of recombinant protein transiently expressed in Nicotiana benthamiana, enhanced green fluorescence protein (EGFP) was expressed as a fusion with 5 or 32 tandem repeats of a serine–proline motif, designated (SP)5 or (SP)32, which is known to direct extensive Hyp-O-glycosylation in plants. EGFP containing the (SP)n motif showed enhanced yields in the order as follows: EGFP<EGFP-(SP)5≪(SP)5-EGFP<(SP)32-EGFP. The EGFP equivalent yield of (SP)32-EGFP was up to 16-fold greater than that of the EGFP control. In addition, both fully glycosylated (SP)32-EGFP (∼115kDa) and partially glycosylated (SP)32-EGFP (∼40kDa) were detected in protein extracts of N. benthamiana. These two types of glycoforms were completely segregated between media and cells in tobacco BY-2 cell cultures.

Quantitative Evaluation of E1 Endoglucanase Recovery from Tobacco Leaves Using the Vacuum Infiltration-Centrifugation Method

As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltrated Nicotiana benthamiana leaves, up to 81% of a truncated version of E1 endoglucanase from Acidothermus cellulolyticus was recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins.

Quantifying the Effects of Frequency and Amplitude of Periodic Oxygen-Related Stress on Recombinant Protein Production in Pichia pastoris

Pichia pastoris is an attractive candidate platform for recombinant protein production. Dissolved oxygen is one of the most important factors in the cultivation of P. pastoris. However, the effect of oxygen on triggering productivity led to ambivalent results. In our earlier work, a two-compartment system, consisting of a single reactor coupled with a plug flow reactor (PFR), has been proposed as a tool to improve protein quantity and quality. The goal of this work was to investigate the effects of frequency (the residence time of broth in the PFR) and amplitude (the dissolved oxygen level in the reactor) of the stress on productivity, titer and physiology. A recombinant P. pastoris strain, which expressed horseradish peroxidase, was used as the model system. Thirteen experiments were performed. Multivariate data analysis was done and the results showed that the residence time did not influence titer, productivity and physiology over the range of residence time studied while dissolved oxygen influenced titer and specific productivity in a quadratic function. In other words, an intermediate level of dissolved oxygen (25%) showed the highest specific productivity and titter, irrespective of the residence time in the PFR. In turn, the variation of the residence time and dissolved oxygen did not influence growth physiology, as quantified in biomass and carbon dioxide yields.

Identification of the pivotal role of glutamate in enhancing insect cell growth using factor analysis

Although the insect cell/baculovirus system is an important expression platform for recombinant protein production, our understanding of insect cell metabolism with respect to enhancing cell growth capability and productivity is still limited. Moreover, different host insect cell lines may have different growth characteristics associated with diverse product yields, which further hampers the elucidation of insect cell metabolism. To address this issue, the growth behaviors and utilization profiles of common metabolites among five cultured insect cell lines (derived from two insect hosts, Spodoptera frugiperda and Spodoptera exigua) were investigated in an attempt to establish a metabolic framework that can interpret the different cell growth behaviors. To analyze the complicated metabolic dataset, factor analysis was introduced to differentiate the crucial metabolic variations among these cells. Factor analysis was used to decompose the metabolic data to obtain the underlying factors with biological meaning that identify glutamate (a metabolic intermediate involved in glutaminolysis) as a key metabolite for insect cell growth. Notably, glutamate was consumed in significant amounts by fast-growing insect cell lines, but it was produced by slow-growing lines. A comparative experiment using cells grown in culture media with and without glutamine (the starting metabolite in glutaminolysis) was conducted to further confirm the pivotal role of glutamate. The factor analysis strategy allowed us to elucidate the underlying structure and inter-correlation between insect cell growth and metabolite utilization to provide some insights into insect cell growth and metabolism, and this strategy can be further extended to large-scale metabolomic analysis.

N-Glycosylation of Plant-produced Recombinant Proteins

Plants are gaining increasingly acceptance as a production platform for recombinant proteins. One reason for this is their ability to carry out posttranslational protein modifications in a similar if not identical way as mammalian cells. The capability of plants to carry out human-like complex glycosylation is well known. Moreover, the targeted manipulation of the plant N-glycosylation pathway allows the production of proteins carrying largely homogeneous, human-type oligosaccharides. These outstanding results have placed plants in a favourable position compared to other eukaryotic expression systems. This review provides a comprehensive summary of the N-glycosylation of plant-produced recombinant proteins, the possible impact of plant-specific N-glycans on the human immune system, and recent advances in engineering the plant N-glycosylation pathway towards the synthesis of (complex) human-type glycan structures, highlighting challenges and achievements in the application of these powerful technologies.

Utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells

Genetically transformed lepidopteran insect cell lines have biotechnological applications as constitutive recombinant protein production platforms and improved hosts for baculovirus-mediated recombinant protein production. Insect cell transformation is often accomplished with a DNA construct(s) encoding a foreign protein(s) under the transcriptional control of a baculovirus immediate early promoter, such as the ie1 promoter. However, the potential utility of increasingly stronger promoters from later baculovirus gene classes, such as delayed early (39K), late (p6.9), and very late (polh), has not been systematically assessed. Hence, we produced DNA constructs encoding secreted alkaline phosphatase (SEAP) under the transcriptional control of each of the four temporally distinct classes of baculovirus promoters, used them to transform insect cells, and compared the levels of SEAP RNA and protein production obtained before and after baculovirus infection. The ie1 construct was the only one that supported SEAP protein production by transformed insect cells prior to baculovirus infection, confirming that only immediate early promoters can be used to isolate transformed insect cells for constitutive recombinant protein production. However, baculovirus infection activated transgene expression by all four classes of baculovirus promoters. After infection, cells transformed with the very late (polh) and late (p6.9) promoter constructs produced the highest levels of SEAP RNA, but only low levels of SEAP protein. Conversely, cells transformed with the immediate early (ie1) and delayed early (39K) promoter constructs produced lower levels of RNA, but equal or higher levels of SEAP protein. Unexpectedly, the 39K promoter construct provided tightly regulated, baculovirus-inducible protein production at higher levels than the later promoter constructs. Thus, this study demonstrated the utility of the 39K promoter for insect cell engineering, particularly when one requires higher levels of effector protein production than obtained with ie1 and/or when constitutive transgene expression adversely impacts host cell fitness and/or genetic stability.

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2′-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems.

CHLOROPLAST GENETIC TOOL FOR THE GREEN MICROALGAE HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE, VOLVOCALES)(1).

At present, there is strong commercial demand for recombinant proteins, such as antigens, antibodies, biopharmaceuticals, and industrial enzymes, which cannot be fulfilled by existing procedures. Thus, an intensive search for alternative models that may provide efficiency, safety, and quality control is being undertaken by a number of laboratories around the world. The chloroplast of the eukaryotic microalgae Haematococcus pluvialis Flotow has arisen as a candidate for a novel expression platform for recombinant protein production. However, there are important drawbacks that need to be resolved before it can become such a system. The most significant of these are chloroplast genome characterizations, and the development of chloroplast transformation vectors based upon specific endogenous promoters and on homologous targeting regions. In this study, we report the identification and characterization of endogenous chloroplast sequences for use as genetic tools for the construction of H. pluvialis specific expression vectors to efficiently transform the chloroplast of this microalga via microprojectile bombardment. As a consequence, H. pluvialis shows promise as a platform for expressing recombinant proteins for biotechnological applications, for instance, the development of oral vaccines for aquaculture.

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.

Production of mouse granulocyte‐macrophage colony‐stimulating factor by gateway technology and transgenic rice cell culture

To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.

Comparative transcriptomic profile analysis of fed-batch cultures expressing different recombinant proteins in Escherichia coli

There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-β), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-β and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression.

Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear whether this is because of few attempts or of limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, >50% expressed at levels sufficient for commercial production. Three expressed at 2%-3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivity achieved demonstrate the utility of Chlamydomonas reinhardtii as a robust platform for human therapeutic protein production.

Micro-algae come of age as a platform for recombinant protein production.

A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins.

Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli

Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or “program” entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and β-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions.

Comparative efficiency of subcellular targeting signals for expression of a toxic protein in sugarcane

Transgenic sugarcane plants (Saccharum hybrid) have been proposed as a production platform for recombinant proteins, including those providing pathogen resistance as well as high value therapeutic proteins. For the in planta production of proteins that are potentially toxic, a careful consideration of subcellular location is required in order to optimise yield and to avoid detrimental interaction with plant cellular processes. In this study, avidin, a glycoprotein that is potentially toxic to cells because of its high affinity to the co-vitamin biotin, was used to test the effectiveness of a range of targeting signals. Accumulation of avidin was directed to the apoplast, endoplasmic reticulum and to the lytic and delta type vacuoles. Although targeting to the delta vacuole resulted in the highest yields of avidin, these plants developed a biotin deficient phenotype, indicating that this targeting was not fully effective in protecting cellular biotin pools. Similar problems were also observed when avidin was retained in the endoplasmic reticulum. When avidin was targeted to the lytic vacuole using the targeting signal from the sugarcane legumain, plants remained phenotypically normal; however, avidin was predominantly detected as a degraded product due to site-specific limited proteolysis in the vacuole. For avidin and other potentially toxic products, this lytic vacuole targeting signal may be useful if stability within this proteolytic environment can be improved.

New medicinal plants for the production of vaccines

During the last decade a large number of biopharmaceuticals has entered the market, mainly protein-based pharmaceuticals like hormones, cytokines, antibodies, and vaccines. Several hundred products are currently in clinical trials and already every fourth approval for genuinely new pharmaceuticals is made for a recombinant product. To meet the increasing demand for recombinant protein production capacity alternative technologies are urgently needed. Transgenic plants have been shown to be a versatile production platform for recombinant proteins and several examples proved plants abilities to provide proper proteins for therapeutic applications. Especially in the case of subunit vaccines plants offer also the potential for the easy generation of pharmaceuticals which could be delivered via the oral route. This review is intended to highlight some exclusive features of this novel production system and point out potential applications.

Insect cell culture for industrial production of recombinant proteins.

Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.