System For Production Of Recombinant Proteins Research Articles

Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site.

Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein production was prepared and the enzyme was purified and crystallized in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resolution and the structure was solved by molecular replacement. However, asulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a critical component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes.

A gel-based method for purification of apolipoprotein A-I from small volumes of plasma

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.

Expression and export: recombinant protein production systems for Aspergillus

Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.

Extraction and purification of human interleukin-10 from transgenic rice seeds

Recombinant protein production system using transgenic rice grain offers many advantages in higher accumulation, preservation, lower production cost, ease of scale up and low risk of contamination by toxic materials. We developed a transgenic rice strain whose seeds accumulate human interleukin (IL)-10, a cytokine that suppresses inflammation-related immune responses. We also developed a method of extracting and purifying IL-10 from rice seeds. A biochemical crosslinking method was used to detect the biologically active noncovalent dimer of IL-10. This method was useful for developing efficient methods of refolding and purification. The purified IL-10 comprised only noncovalent dimers and showed higher activity than the commercial IL-10. The purified IL-10 had very low endotoxin contamination and is expected to have broad clinical application.

Learning to predict expression efficacy of vectors in recombinant protein production

BackgroundRecombinant protein production is a useful biotechnology to produce a large quantity of highly soluble proteins. Currently, the most widely used production system is to fuse a target protein into different vectors in Escherichia coli (E. coli). However, the production efficacy of different vectors varies for different target proteins. Trial-and-error is still the common practice to find out the efficacy of a vector for a given target protein. Previous studies are limited in that they assumed that proteins would be over-expressed and focused only on the solubility of expressed proteins. In fact, many pairings of vectors and proteins result in no expression.ResultsIn this study, we applied machine learning to train prediction models to predict whether a pairing of vector-protein will express or not express in E. coli. For expressed cases, the models further predict whether the expressed proteins would be soluble. We collected a set of real cases from the clients of our recombinant protein production core facility, where six different vectors were designed and studied. This set of cases is used in both training and evaluation of our models. We evaluate three different models based on the support vector machines (SVM) and their ensembles. Unlike many previous works, these models consider the sequence of the target protein as well as the sequence of the whole fusion vector as the features. We show that a model that classifies a case into one of the three classes (no expression, inclusion body and soluble) outperforms a model that considers the nested structure of the three classes, while a model that can take advantage of the hierarchical structure of the three classes performs slight worse but comparably to the best model. Meanwhile, compared to previous works, we show that the prediction accuracy of our best method still performs the best. Lastly, we briefly present two methods to use the trained model in the design of the recombinant protein production systems to improve the chance of high soluble protein production.ConclusionIn this paper, we show that a machine learning approach to the prediction of the efficacy of a vector for a target protein in a recombinant protein production system is promising and may compliment traditional knowledge-driven study of the efficacy. We will release our program to share with other labs in the public domain when this paper is published.

The Effect of Photoperiod on .BETA.-glucuronidase Gene Expression under Control CaMV-35S Promoter in Transgenic Lettuce

We investigated the effects of different photoperiods on the expression level of GUS genes regulated by the CaMV-35S promoter in leaf lettuce (Lactuca sativa L. cv. ‘Greenwave’) for a recombinant protein production system. A leaf disk experiment and a cultivation experiment were conducted in different photoperiods. A linear relationship (R2=0.9934) was observed between the lengths of photoperiods and GUS activities per unit fresh weight in the leaf disk experiment. This result indicates that the expression of the CaMV-35S promoter during a photoperiod was much more enhanced than that during a dark period. In the cultivation experiment, a longer photoperiod helped not only to enhance the growth of a lettuce plant but also to increase its GUS activity. As a result, GUS activity per plant under the 24:0 h photoperiod was significantly (P‹0.05) higher than that under the 16:8 and 8:16 h photoperiods. However, the question remains whether these results for GUS-CaMV-35S promoter can be applied to other recombinant proteins or promoters. Further studies are thus required to establish a practical strategy for designing an efficient recombinant production system in a plant factory.

Identification and characterization of Rhipicephalus (Boophilus) microplus candidate protective antigens for the control of cattle tick infestations

The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant Rhipicephalus microplus Bm86 antigen has been shown to protect cattle against tick infestations. However, variable efficacy of Bm86-based vaccines against geographic tick strains has encouraged the research for additional tick-protective antigens. Herein, we describe the analysis of R. microplus glutathione-S transferase, ubiquitin (UBQ), selenoprotein W, elongation factor-1 alpha, and subolesin (SUB) complementary DNAs (cDNAs) by RNA interference (RNAi) in R. microplus and Rhipicephalus annulatus. Candidate protective antigens were selected for vaccination experiments based on the effect of gene knockdown on tick mortality, feeding, and fertility. Two cDNA clones encoding for UBQ and SUB were used for cattle vaccination and infestation with R. microplus and R. annulatus. Control groups were immunized with recombinant Bm86 or adjuvant/saline. The highest vaccine efficacy for the control of tick infestations was obtained for Bm86. Although with low immunogenic response, the results with the SUB vaccine encourage further investigations on the use of recombinant subolesin alone or in combination with other antigens for the control of cattle tick infestations. The UBQ peptide showed low immunogenicity, and the results of the vaccination trial were inconclusive to assess the protective efficacy of this antigen. These experiments showed that RNAi could be used for the selection of candidate tick-protective antigens. However, vaccination trials are necessary to evaluate the effect of recombinant antigens in the control of tick infestations, a process that requires efficient recombinant protein production and formulation systems.

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori

To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4-Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)-EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 microg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 microg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.

Xplor® 2—an optimized transformation/expression system for recombinant protein production in the yeast Arxula adeninivorans

Combining ease of genetic manipulation and fermentation with the ability to secrete and to glycosylate proteins in the basic eukaryotic manner, Arxula adeninivorans provides an attractive expression platform. Based on a redesign of the basic vector, a new Arxula vector system, Xplor 2, for heterologous gene expression was established, which allows (1) the construction of expression plasmids for supertransformation of A. adeninivorans strains secreting target proteins of biotechnological interest and (2) the integration of small vector cassettes consisting of yeast DNA sequences only. For this purpose, a set of modules including the ATRP1m selection-marker module, expression modules for constitutive expression of the genes phyK (Klebsiella-derived phytase) and IFNalpha2a (human interferon alpha), the HARS (Hansenula polymorpha autonomous replication sequence) for autonomous replication and the chaperone module AHSB4 promoter -HpCNE1 gene (calnexin) -PHO5 terminator to improve secretion efficiency were constructed and integrated in various combinations in the basic vector Xplor 2. After removal of the complete Escherichia coli-based plasmid parts (resistance marker, ColE1 ori and f1(-) origin), the remaining yeast-based linear vector fragment with or without rDNA targeting sequences were transformed as yeast rDNA integrative expression cassettes and yeast integrative expression cassettes (YICs), respectively, and the resulting strains were tested for their capacity to secrete PhyK or IFNalpha2a. Maximal expression levels were consistently obtained using YICs for transformation irrespective of whether or not they carry HARS and/or calnexin modules. It is recommended that at least 50 such transformants be analyzed to ensure selection of the best transformants.

Parallel gene cloning and protein production in multiple expression systems

To quickly find an optimal expression system for recombinant protein production, a set of vectors with the same restriction sites were constructed for parallel cloning of a target gene and recombinant protein production in prokaryotic and eukaryotic expression systems, simultaneously. These vectors include nucleotide sequences encoding protein tags and protease recognition sites for tag removal, followed by the cloning sites 5'-EcoRI/3'-XhoI identical in these vectors for ligating with the sticky-end PCR product of a target gene. Our vectors allow parallel gene cloning and protein production in multiple expression systems with minimal cloning effort.

Study of kinetic parameters for the production of recombinant rabies virus glycoprotein

Gene expression in insect cells is an advantageous system for recombinant protein production, mainly because of its capacity to produce complex proteins with correct post-translational modifications. Recently, we identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of rabies virus glycoprotein, expressed in Drosophila melanogaster cells, by about 60%. In this work, the kinetic parameters for cell growth and recombinant rabies virus glycoprotein production were determined in cultures of transfected Drosophila melanogaster Schneider 2 (S2) cells expressing recombinant rabies virus glycoprotein (rRVGP), enriched and non-enriched with the hemolymph of Lonomia obliqua (Hb). The highest concentration of rRVGP was achieved at the beginning of the culture enriched with Hb, indicating that the cells produce greater amounts of rRVGP per cell (specific rRVGP concentration) at the early exponential growth phase. After day 8, a decrease in the concentration of rRVGP (ng/mL) was observed, probably due to protein decomposition. The average specific rRVGP production rate (mu(rRVGP)) was 30 ng rRVGP/10(7)cell.day, higher than that observed in the non-enriched culture.

Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco

Rhizosecretion is an attractive technology for the production of recombinant proteins from transgenic plants. However, to date, yields of plant-derived recombinant pharmaceuticals by this method have been too low for commercial viability. Studies conducted focused on three transgenic plant lines grown in hydroponic culture medium, two expressing monoclonal antibodies Guy's 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin-N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator alpha-naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 microg/g root dry weight/24 h for Guy's 13, 10.43 microg/g root dry weight/24 h for 4E10, and 766 microg/g root dry weight/24 h for cyanovirin-N, the highest figures so far reported for a full-length antibody and a recombinant protein, respectively. The plant growth regulators indole-butyric acid, 6-benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guy's 13. The effect of the growth regulators differed, as alpha-naphthalene acetic acid and indole-butyric acid increased the root dry weight of hydroponic plants, whereas the cytokinins benzylaminopurine and kinetin increased rhizosecretion without affecting root mass. A comparative glycosylation analysis between MAb Guy's 13 purified from either hydroponic culture medium or from leaf extracts demonstrated a similar pattern of glycosylation comprising high mannose to complex glycoforms. Analysis of the hydroponic culture medium at harvest revealed significantly lower and less complex levels of proteolytic enzymes, in comparison with leaf extracts, which translated to a higher proportion of intact Guy's 13 IgG in relation to other IgG products. Hydroponic medium could be added directly to a chromatography column for affinity purification, allowing simple and rapid production of high purity Guy's 13 antibody. In addition to the attractiveness of controlled cultivation within a contained environment for pharmaceutical-producing plants, this study demonstrates advantages with respect to the quality and downstream purification of recombinant proteins.

A Dynamic Model for Simulating Recombinant Protein Production in Transgenic Lettuce for Optimum Environmental Control

A dynamic model for simulating recombinant protein production in transgenic lettuce for optimum environmental control was developed. The model comprised three main parameters, which were leaf fresh weight, the number of cells, and amount of protein in each leaf. The differential equations of the model were integrated using the modeling software STELLATM. The model was calibrated based on the measured data of transgenic lettuce plants which were grown in a plant factory for 50 days from seeding. The plants contained an introduced Cauliflower mosaic virus-35S promoter fused onto the β-glucuronidase gene. Simulations were conducted to investigate the effect of plant growth control on the productivity of recombinant proteins which have various stabilities. The simulations indicated the importance of recombinant protein stability. If the parameters corresponding to the production and degradation of a recombinant protein can be identified, the simulation will be a strong tool for devising an environmental control strategy for highly efficient recombinant protein production systems.

Production of a His‐tagged canecystatin in transgenic sugarcane and subsequent purification

Transgenic plants have been used widely as expression systems of recombinant proteins in recent years. This process can be an efficient alternative for the large-scale production of proteins. In this work, we present the establishment of transgenic sugarcane expressing a His-tagged canecystatin under the control of the maize ubiquitin promoter. A number of studies have demonstrated that cystatins, which are natural inhibitors of cysteine proteinases, can be used for protection against insect attacks. A transformed sugarcane plant that presented high levels of (HIS)CaneCPI-1 expression, was selected for the purification of this protein through affinity chromatography in a nickel column. This purified (HIS)CaneCPI-1 was immunodetected using a polyclonal antibody, which was also able to detect the (HIS)CaneCPI-1 in a crude extract from transgenic plant leaves. Assays of inhibitory activity performed with the purified (HIS)CaneCPI-1 revealed its ability to inhibit the catalytic activity of midgut cysteine proteinase partially purified from the sugarcane weevil Sphenophorus levis and human cathepsin L in nanomolar order. These studies demonstrate that sugarcane is a viable expression system for recombinant protein production.

Biopharmaceuticals from Plants: A Multitude of Options for Posttranslational Modifications

In 1982 the first recombinant therapeutic, human insulin, was introduced into the market and started a new branch of pharmaceutical development, manufacture, and therapy options. To date, more than 130 recombinant protein therapeutics have been approved by the US Food and Drug Administration (FDA) and many more are being developed world wide. With the increasing number of protein therapeutics the number of potential production organisms is also expanding, and posttranslational modification of proteins has become a topic of special focus. One major difference between small-molecule drugs and protein therapeutics is that the latter are reliant on a host organism for their production and this can have a large influence on the final structure and can ultimately affect the pharmacokinetics, immunogenicity, and the function of the protein depending on the production process. Plants can be efficiently used as production systems for recombinant proteins thereby offering a variety of options for transgene targeting and modification. This review is intended to give an overview about the potential of plants to serve as a production system for therapeutic and prophylactic biopharmaceuticals with respect to posttranslational modifications.

Moss bioreactors producing improved biopharmaceuticals

Plants may serve as superior production systems for complex recombinant pharmaceuticals. Current strategies for improving plant-based systems include the development of large-scale production facilities as well as the optimisation of protein modifications. While post-translational modifications of plant proteins generally resemble those of mammalian proteins, certain plant-specific protein-linked sugars are immunogenic in humans, a fact that restricts the use of plants in biopharmaceutical production so far. The moss Physcomitrella patens was developed as a contained tissue culture system for recombinant protein production in photo-bioreactors. By targeted gene replacements, moss strains were created with non-immunogenic humanised glycan patterns. These were proven to be superior to currently used mammalian cell lines in producing antibodies with enhanced effectiveness.

Multiplexed expression and screening for recombinant protein production in mammalian cells.

BackgroundA variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.ResultsA nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale.ConclusionThe combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful for applications such as the generation of receptor protein microarrays.

High cell density fed batch and perfusion processes for stable non‐viral expression of secreted alkaline phosphatase (SEAP) using insect cells: Comparison to a batch Sf‐9‐BEV system

The development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening. The expression of the Sf-9SEAP cells was compared with the BEVS system. It was observed that, the yield obtained in BEVS was similar to the batch Sf-9SEAP at 8 and 7 IU/mL, respectively. The productivity of this foreign gene product with the stable cells was enhanced by bioprocess intensification employing the fed-batch and perfusion modes of culture to increase the cell density in culture. The fed batch process yielded a maximum cell density of 28 x 10(6) cells/mL and 12 IU/mL of SEAP. Further improvements in the productivity could be made using the perfusion process, which demonstrated a stable production rate for extended periods of time. The process was maintained for 43 days, with a steady-state cell density of 17-20 x 10(6) cells/mL and 7 IU/mL SEAP. The total yield obtained in the perfusion process (394 IU) was approximately 22 and 8 times higher than that obtained in a batch (17.6 IU) and fed batch (46.1 IU) process, respectively.

Downstream process engineering evaluation of transgenic soybean seeds as host for recombinant protein production

The advantages of using seeds for the production of recombinant proteins with plant-based expression system has been demonstrated by several researchers. The high productivity makes soybean a potential system for large-scale recombinant protein production. However, there is a lack of detailed engineering studies of the downstream process (DSP) of recombinant proteins produced in transgenic soybean. In this work, we evaluated the use of transgenic soybean seeds as hosts for the production of recombinant proteins from a downstream process (DSP) engineering standpoint. Recombinant β-glucuronidase (rGUS), was used as a model for extraction and purification studies. This study showed, that even a protein with acidic p I (rGUS) can be successfully separated from native soybean proteins, which also have acidic p I. Maximum GUS specific activity (9.5 × 10 3 U/mg) with high total activity recovery (8.9 × 10 4 U/mL) was obtained using a simple extraction solution composed of 50 mmol/L citrate buffer at pH 5.25. Purification of rGUS was evaluated by a two-step chromatographic procedure – anion-exchange followed by hydrophobic interaction chromatography – which was compared to the purification of rGUS from transgenic corn and canola. Overall purification factor and activity recovery obtained were 97.3 and 110% (a value higher than 100% probably due to removal of an inhibitor). Comparison of this study with similar ones made with corn and canola seeds indicates that in terms of DSP soybean seeds can be considered a potentially viable plant system for the production of recombinant proteins.

Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization

Type I secretion system (TISS) of Gram-negative bacteria permits proteins to be secreted directly from the cytoplasm to the external medium by a single, energy-coupled step. To examine whether this system can be used as an extracellular production system of recombinant proteins, Escherichia coli alkaline phosphatase (AP) was fused to a C-terminal region of Pseudomonas sp. MIS38 lipase (PML) and examined for secretion using the E.coli cells carrying the heterologous TISS. PML is one of the passenger proteins of TISS and contains 12 repetitive sequences and a secretion signal at the C-terminal region. The fusion protein was efficiently secreted to the extracellular medium, while AP was not secreted at all, indicating that the secretion of AP is promoted by a secretion signal of PML. The repetitive sequences were not so important for secretion of the fusion protein, because the secretion level of the fusion protein containing entire repeats ( approximately 10 mg/l culture) was only 2-fold higher than that of the fusion protein without repeats. The fusion protein purified from the culture supernatant existed as a homodimer, like AP, and was indistinguishable from AP in enzymatic properties and stability.