High Levels Of Recombinant Proteins Research Articles

Non-target Effects of Hyperthermostable α-Amylase Transgenic Nicotiana tabacum in the Laboratory and the Field.

Thermostable α-amylases are important enzymes used in many industrial processes. The expression of recombinant Pyrococcus furiosus α-amylase (PFA) in Nicotiana tabacum has led to the accumulation of high levels of recombinant protein in transgenic plants. The initial steps to registering the transgenic tobacco at a commercial production scale and growing it in the field requires a risk assessment of potential non-target effects. The objective of this study was to assess the effect of feeding on transgenic tobacco with 2 indigenous insect species commonly associated with wild and commercial tobacco involving plants grown and evaluated under laboratory and field conditions. The highest levels of PFA ranged from 1.3 to 2.7 g/kg leaf fresh weight produced in the field-grown cultivars Con Havana and Little Crittenden, respectively. These two cultivars also had the highest nicotine (ranging from 4.6 to 10.9 mg/g), but there was little to no negative effect for either tobacco hornworm Manduca sexta L. or aphid Myzus nicotianae (Blackman). Both laboratory and field trials determined no short term (5 days) decrease in the survival or fecundity of the tobacco aphid after feeding on PFA transgenic tobacco compared to non-transgenic plants. In the field, tobacco hornworm larvae showed no differences in survival, final larval weights or development time to adult stage between transgenic lines of four cultivars and their corresponding wild type controls. Laboratory studies confirmed the field trial results indicating the low risk association of PFA expressed in tobacco leaves with tobacco hornworms and aphids that would feed on the transgenic plants.

Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts.

High accumulation of heterologous proteins expressed from the plastid genome has sometimes been reported to result in compromised plant phenotypes. Comparisons of transplastomic plants to wild-type (WT) are typically made in environmentally controlled chambers with relatively low light; little is known about the performance of such plants under field conditions. Here, we report on two plastid-engineered tobacco lines expressing the bacterial cellulase Cel6A. Field-grown plants producing Cel6A at ~20% of total soluble protein exhibit no loss in biomass or Rubisco content and only minor reductions in photosynthesis compared to WT. These experiments demonstrate that, when grown in the field, tobacco possesses sufficient metabolic flexibility to accommodate high levels of recombinant protein by increasing total protein synthesis and accumulation and/or by reallocating unneeded endogenous proteins. Based on current tobacco cultivation practices and readily achievable recombinant protein yields, we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods.

BacMam System for Rapid Recombinant Protein Expression in Mammalian Cells.

Baculovirus expression vector system (BEVS) is an established technology for recombinant protein expression in insect cells. Further, BEVS-mediated gene transduction of mammalian cells (BacMam) is emerging as a technique for high level recombinant protein expression in mammalian cells. Here, we describe generic method in using BEVS as a BacMam for rapid recombinant protein expression in mammalian cells.

Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system.

Bacillus amyloliquefaciens K11 is a hyperproducer of extracellular neutral protease, which can produce recombinant homologous protein steadily and is amenable to scale up to high-cell density fermentation. The present study aims to genetically modify strain K11 as a highly efficient secretory expression system for high-level production of heterologous proteins. Using B. amyloliquefaciens K11 and alkaline protease gene BcaprE as the expression host and model gene, the gene expression levels mediated by combinations of promoters PamyQ, PaprE and Pnpr and signal peptides SPamyQ, SPaprE and SPnpr were assessed on shake flask level. The PamyQ-SPaprE was found to be the best secretory expression cassette, giving the highest enzyme activities of extracellular BcaprE (13,800 ± 308U/mL). Using the same expression system, the maltogenic α-amylase Gs-MAase and neutral protease BaNPR were successfully produced with the enzyme activities of 19. ± 0.2U/mL and 17,495 ± 417U/mL, respectively. After knocking out the endogenous neutral protease-encoding gene Banpr, the enzyme activities of BcaprE and Gs-MAase were further improved by 25.4% and 19.4%, respectively. Moreover, the enzyme activities of BcaprE were further improved to 30,200 ± 312U/mL in a 15L fermenter following optimization of the fermentation conditions. In the present study, the genetically engineered B. amyloliquefaciens strain 7-6 containing PamyQ-SPaprE as the secretory expression cassette was developed. This efficient expression system shows general applicability and represents an excellent industrial strain for the production of heterologous proteins.

Optimizing host cell physiology and stress avoidance for the production of recombinant human tumour necrosis factor α in Escherichia coli.

As high-level recombinant protein production (RPP) exerts a massive stress on the production host, an extensive literature on RPP optimization focuses on separating the growth phase from RPP production once sufficient biomass has been obtained. The aim of the current investigation was to optimize the benefits of the relatively neglected alternative strategy to achieve high-level RPP during growth by minimizing stress on the host. High yields of the biopharmaceutical recombinant human tumour necrosis factor alpha (rhTNFα) were obtained by fed-batch fermentation relevant to industrial production based upon parameters that most severely affected RPP in preliminary laboratory scale batch cultures. Decreasing the inducer concentration and growth temperature, but increasing the production period, were far more effective for increasing RPP yields than changing the growth phase at which production was induced. High yields of up to 5 g l-1 of rhTNFα were obtained with minimal plasmid loss, even in synthetic media that lack animal-derived components and are therefore fully compliant with regulatory requirements. Most of the product was soluble and biologically active. In summary, stress minimization was shown to be an effective way to optimize the production of rhTNFα. Data generated in shake-flask experiments allowed the design of intensified bioreactor cultures in which RPP and growth could be balanced, leading to higher yield of both rhTNFα and biomass than with previous fermentations. An additional benefit of this approach is avoidance of lysis during harvesting and downstream processing.

Engineering of an Episomal Plasmid Suitable for High-Throughput Expression in Pichia pastoris.

This study describes the design and evaluation of an expression vector for Pichia pastoris (pPICZαBHF), which is based on the commercial vector construct pPICZαB. The performance of pPICZαBHF was evaluated with red fluorescent protein as a reporter. Additional His- and Flag-tags on the N-terminal ensured a simplified protein purification procedure. Transformation efficiency, expression level and plasmid maintenance were studied in order to test the functionality and usefulness of the constructed vector. We found that high transformation efficiencies were achieved using pPICZαBHF plasmid for yeast cell transformation in comparison with the commercial vector pPICZαB, which has to be integrated into the Pichia genome. However, expression levels of the recombinant protein were generally lower compared to the commercial construct. Recombinant plasmids were shown to be maintained in cells for approximately five days. Although pPICZαBHF may not be suitable for the production of high levels of recombinant protein, the simplicity of this P. pastoris expression system may still be of interest for the expression of proteins involved in cofactor regeneration or the expression of reporter genes. In addition, high transformation efficiency of pPICZαBHF may be beneficial for the applications such as high-throughput screening of mutant gene libraries.

Metabolic phenotyping of CHO cells varying in cellular biomass accumulation and maintenance during fed-batch culture.

CHO cell lines capable of high-level recombinant protein product biosynthesis during fed-batch culture are still generally obtained by intensive empirical screening of transfected cells rather than knowledge-guided cellular engineering. In this study, we investigate how CHO cell lines create and maintain cellular biosynthetic capacity during fed-batch culture to achieve the optimal combination of rapid exponential proliferation and extended maintenance of high cell biomass concentration. We perform a comparative meta-analysis of mitochondrial and glycolytic functions of 22 discrete parental CHO cell lineages varying in fed-batch culture performance to test the hypotheses that (i) "biomass-intensive" CHO cells exhibit conserved differences in metabolic programming and (ii) it is possible to isolate parental CHO cell lines with a biomass-intensive phenotype to support fed-batch bioproduction processes. We show that for most parental CHO cell lines, rapid proliferation and high late-stage culture performance are mutually exclusive objectives. However, quantitative dissection of mitochondrial and glycolytic functions revealed that a small proportion of clones utilize a conserved metabolic program that significantly enhances cellular glycolytic and mitochondrial oxidative capacity at the onset of late-stage culture. We reveal the central importance of dynamic metabolic re-programming to activate oxidative mitochondrial function as a necessary mechanism to support CHO cell biosynthetic performance during culture.

Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHOBRI/rcTA) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHOBRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHOBRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins.

A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production.

Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usually necessitates a time intense screening step in order to find strains with the desired productivity. Some of the factors causing this clonal variability can be overcome using episomal vectors containing an autonomously replicating sequence (ARS). Here, we report on the discovery, characterization, and application of a fragment of mitochondrial DNA from P. pastoris for use as an ARS. First encountered as an off-target event in an experiment aiming for genomic integration, the newly created circular plasmid named “pMito” consists of the expression cassette and a fragment of mitochondrial DNA. Multiple matches to known ARS consensus sequence motifs, but no exact match to known chromosomal ARS from P. pastoris were detected on the fragment, indicating the presence of a novel ARS element. Different variants of pMito were successfully used for transformation and their productivity characteristics were assayed. All analyzed clones displayed a highly uniform expression level, exceeding by up to fourfold that of a reference with a single copy integrated in its genome. Expressed GFP could be localized exclusively to the cytoplasm via super-resolution fluorescence microscopy, indicating that pMito is present in the nucleus. While expression levels were homogenous among pMito clones, an apparent upper limit of expression was visible that could not be explained based on the gene dosage. Further investigation is necessary to fully understand the bottle-neck hindering this and other ARS vectors in P. pastoris from reaching their full capability. Lastly, we could demonstrate that the mitochondrial ARS from P. pastoris is also suitable for episomal vector transformation in Saccharomyces cerevisiae, widening the potential for biotechnological application. pMito displayed strong potential to reduce clonal variability in experiments targeting recombinant protein production. These findings also showcase the as of yet largely untapped potential of mitochondrial ARS from different yeasts for biotechnological applications.

Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins.

BackgroundGene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression.ResultsHere, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins.ConclusionsAn automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0316-3) contains supplementary material, which is available to authorized users.

Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production.

Membrane proteins perform critical cellular functions in all living organisms and constitute major targets for drug discovery. Escherichia coli has been the most popular overexpression host for membrane protein biochemical/structural studies. Bacterial production of recombinant membrane proteins, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low final biomass and minute volumetric yields. In this work, we aimed to rewire the E.coli protein-producing machinery to withstand the toxicity caused by membrane protein overexpression in order to generate engineered bacterial strains with the ability to achieve high-level membrane protein production. To achieve this, we searched for bacterial genes whose coexpression can suppress membrane protein-induced toxicity and identified two highly potent effectors: the membrane-bound DnaK cochaperone DjlA, and the inhibitor of the mRNA-degrading activity of the E.coli RNase E, RraA. E.coli strains coexpressing either djlA or rraA, termed SuptoxD and SuptoxR, respectively, accumulated markedly higher levels of final biomass and produced dramatically enhanced yields for a variety of prokaryotic and eukaryotic recombinant membrane proteins. In all tested cases, either SuptoxD, or SuptoxR, or both, outperformed the capabilities of commercial strains frequently utilized for recombinant membrane protein production purposes.

Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.

Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production.

Transient Protein Expression by Agroinfiltration in Lettuce.

Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level.

Transient Expression of Mammalian Genes in N. benthamiana to Modulate N-Glycosylation.

Nicotiana benthamiana has shown great success as a platform for the production of recombinant proteins. Here, we describe methods to transiently express high levels of recombinant proteins and simultaneously modulate their glycosylation pattern toward human-like structures. The method aims to generate recombinant proteins with a targeted largely homogeneous glycosylation profile for structure-function studies.

High-level recombinant protein production by the basidiomycetous yeast Pseudozyma antarctica under a xylose-inducible xylanase promoter.

Yeast host-vector systems are useful tools for the production of recombinant proteins. Here, we report the construction of a new high-level expression plasmid pPAX1-neo for the basidiomycetous yeast, Pseudozyma antarctica. pPAX1-neo harbours a xylose-inducible expression cassette under control of the xylanase promoter and terminator of P. antarctica T-34, a selection cassette of neomycin/G418 with an Escherichia coli neomycin resistance gene under control of the homocitrate synthase promoter of strain T-34, and an autonomously replicating sequence fragment of Ustilago maydis (UARS). Biodegradable plastic (BP)-degrading enzymes of P. antarctica JCM10317 (PaE) and Paraphoma-related fungal strain B47-9 (PCLE) were used as reporter proteins and inserted into pPAX1-neo, resulting in pPAX1-neo::PaCLE1 and pPAX1-neo::PCLE, respectively. Homologous and heterologous BP-degrading enzyme production of transformants of P. antarctica T-34 were detected on agar plates containing xylose and emulsified BP. Recombinant PaE were also produced by transformants of other Pseudozyma strains including Pseudozyma aphidis, Pseudozyma rugulosa, and Pseudozyma tsukubaensis. To improve the stability of transformed genes in cells, the UARS fragment was removed from linearized pPAX1-neo::PaCLE1 and integrated into the chromosome of the P. antarctica strain, GB-4(0), which was selected as a PaE producer in xylose media. Two transformants, GB-4(0)-X14 and X49, had an 11-fold higher activity compared with the wild type strain in xylose-containing liquid media. By xylose fed-batch cultivation using a 3-L jar fermentor, GB-4(0)-X14 produced 73.5 U mL(-1) of PaE, which is 13.4-fold higher than that of the wild type strain GB-4(0), which produced 5.5 U mL(-1) of PaE.

High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization.

The major platform for high level recombinant protein production is based on genetically modified microorganisms like Escherichia coli (E. coli) due to its short dividing time, ability to use inexpensive substrates and additionally, its genetics is comparatively simple, well characterized and can be manipulated easily. Here, we investigated the possibilities of finding the best media for high cell density fermentation, by analyzing different media samples, focusing on improving fermentation techniques and recombinant protein production. Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria–Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively). However, in fed-batch fermentation processes when using MMM after 25 h cultivation, it was possible to yield an optical density (OD600) of 139 corresponding to 172 g/L of wet biomass was produced in a 30 L TV Techfors-S Infors HT fermenter, with a computer controlled nutrient supply (glucose as a carbon source) delivery system, indicating nearly 1.5 times that obtained from TB. Upon purification, a total of 1.65 mg/g of protein per gram cell biomass was obtained and the purified AviPure showed affinity for immunoglobulin. High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition. It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0155-y) contains supplementary material, which is available to authorized users.

Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells.

Background:Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis.Objectives:The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine.Materials and Methods:Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB).Results:The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods.Conclusions:The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites and are promising approaches for antigen preparation in vaccine development.

Complete Genome Sequence of Escherichia coli Strain BL21.

Escherichia coli strain BL21 is one of the widely used bacterial hosts for high-level recombinant protein production and for other applications. Here, we present the complete genome sequence of a commercial version of the Escherichia coli BL21 strain.

Transient Glyco-Engineering of N. benthamiana Aiming at the Synthesis of Multi-antennary Sialylated Proteins.

Recombinantly produced therapeutic proteins bring benefits to patients and production companies alike. However, due to high production costs the potential of this technology cannot be fully tapped and therefor safe, scalable, and economic alternatives to the standard mammalian cell culture-based manufacturing systems are needed. Plant-based expression systems with their current technological advances constitute such an alternative. Many recombinant biopharmaceuticals are glycoproteins and their structural properties and pharmacokinetics are strongly influenced by their glycosylation profile. Differences in glycosylation between plants and mammals can for this reason result in different therapeutic efficacies. In particular, low levels of sialylation may lead to a short serum half-life of therapeutic proteins and nonhuman types of glycosylation can induce degradation and immunogenic responses. Controlling glycosylation of plant-derived therapeutics is therefore fundamental to enhance their efficacy and eliminate possible adverse effects caused by non-authentic glycosylation. Here we describe methods to transiently express high levels of recombinant proteins in Nicotiana benthamiana and simultaneously modulate their glycosylation pattern towards the synthesis of highly sialylated humanlike structures.

Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1.

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.