Recombinant Green Fluorescent Protein Research Articles

Soft sensor control of metabolic fluxes in a recombinant Escherichia coli fed-batch cultivation producing green fluorescence protein

A soft sensor approach is described for controlling metabolic overflow from mixed-acid fermentation and glucose overflow metabolism in a fed-batch cultivation for production of recombinant green fluorescence protein (GFP) in Escherichia coli. The hardware part of the sensor consisted of a near-infrared in situ probe that monitored the E. coli biomass and an HPLC analyzer equipped with a filtration unit that measured the overflow metabolites. The computational part of the soft sensor used basic kinetic equations and summations for estimation of specific rates and total metabolite concentrations. Two control strategies for media feeding of the fed-batch cultivation were evaluated: (1) controlling the specific rates of overflow metabolism and mixed-acid fermentation metabolites at a fixed pre-set target values, and (2) controlling the concentration of the sum of these metabolites at a set level. The results indicate that the latter strategy was more efficient for maintaining a high titer and low variability of the produced recombinant GFP protein.

Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction

BackgroundRecycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ). We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD)1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs.MethodsLocalization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1−/− mouse skeletal muscle.ResultsEndogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1−/− mouse skeletal muscle than in wild-type skeletal muscle.ConclusionEHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1−/− muscle may be due to functional compensation by other EHD paralogs.

Protective Effects of Mesenchymal Stem Cells with Transient Overexpression of Hmgb1 on Balloon-Induced Carotid Artery Injury

Mesenchymal stem cells (MSC) play a crucial role in endothelial repair after artery injury. The high mobility group box 1 (HMGB1) is a key modulator of the homing of MSC to impaired artery and endothelialization. This study was aimed to determine whether balloon-induced carotid artery injury could be improved by transplantation with MSC modified by HMGB1. MSC were infected by adenoviral serotype 5 encoding recombinant green fluorescent protein (GFP) gene and HMGB1 (ad5GFP-HMGB1). The expression of HMGB1, vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) was detected in MSC using Real-time PCR, Western blot and semi-quantitative immunohistochemical assays. In vivo, reendothelialization was examined in rats subjected to carotid artery injury. The homing of MSC was observed under fluorescence microscopy, and the levels of serum tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) was assessed by ELISA assay. As a result, compared with the MSC group, the expression of HMGB1, VEGF and PCNA was markedly increased, vascular reendothelialization was accelerated, and the levels of serum TNF-α and CRP were decreased in group ad5GFP and ad5GFP-HMGB1. Transplantation of MSC infected with adGFP-HMGB1 strengthened the MSC effect. Taken together, modification of HMGB1 can enhance the protective effects of MSC on balloon-induced carotid artery injury through up-regulation of VEGF and PCNA expression and inhibition of the inflammatory response. HMGB1 in MSC may represent a novel therapeutic target for the treatment of endothelial repair.

Chemical modification of Nitzschia panduriformis's frustules for protein and viral nanoparticle adsorption

The frustule of diatoms, through appropriate chemical modification, can be developed for a high adsorption level of recombinant proteins and viral nanoparticles. Field emission scanning electron microscopy (FE-SEM) analysis of clean frustules revealed a 3D loculate areolae structure (valvar phase porous pattern of the siliceous cell wall). Isocyanatopropyl triethoxysilane (IPS) and iminodiacetic acid (IDA) were used to immobilize Cu2+ ions (an average Cu2+ adsorption capacity about 190μmol of Cu2+/ml of the Cu2+-coupled biosilica reached). FE-SEM, energy dispersion X-ray spectroscopy (EDS) and Fourier transform infrared (FT-IR) were used to confirm the chemical modification of the Cu2+-coupled biosilica. Protein adsorption was confirmed with the detection of a recombinant (His)6-tagged green fluorescent protein binding using fluorescent microscopy. Infectious bursal disease virus VP2-441 subviral particles (SVPs) were found to bind to the Cu2+-coupled biosilica (approximately 3×10−9mol of VP2-441 SVPs/ml of modified frustules), a level higher than the previously obtained 9×10−10mol/ml for SVP binding using a commercial Ni–NTA resin. These give diatom frustules the potential to be developed into a material useful in viral nanoparticle purification systems or as a biosensor for the detection of viruses.

Functional heterogeneity and heritability in CHO cell populations

In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing--new parental cell lines which are inherently more "fit for purpose." One-hundred and ninety-nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell-specific proliferation rate during extended deep-well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell-specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub-culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N-glycan processing were identified. Finally, for clonal populations most "evolved" by extended sub-culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N-glycosylation. Rapid-specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone-specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and isolate CHO cell variants with improved functional properties in vitro.

Recombinant Interferon-γ Lentivirus Co-Infection Inhibits Adenovirus Replication Ex Vivo

Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P = 0.005–0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P = 0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

Autophagy in the Thymic Epithelium Is Dispensable for the Development of Self-Tolerance in a Novel Mouse Model

The thymic epithelium plays critical roles in the positive and negative selection of T cells. Recently, it was proposed that autophagy in thymic epithelial cells is essential for the induction of T cell tolerance to self antigens and thus for the prevention of autoimmune diseases. Here we have tested this hypothesis using mouse models in which autophagy was blocked specifically in epithelial cells expressing keratin 14 (K14), including the precursor of thymic epithelial cells. While the thymic epithelial cells of mice carrying the floxed Atg7 gene (ATG7 f/f) showed a high level of autophagy, as determined by LC3 Western blot analysis and fluorescence detection of the recombinant green fluorescent protein (GFP)-LC3 reporter protein on autophagosomes, autophagy in the thymic epithelium was efficiently suppressed by deletion of the Atg7 gene using the Cre-loxP system (ATG7 f/f K14-Cre). Suppression of autophagy led to the massive accumulation of p62/sequestosome 1 (SQSTM1) in thymic epithelial cells. However, the structure of the thymic epithelium as well as the organization and the size of the thymus were not altered in mutant mice. The ratio of CD4 to CD8-positive T cells, as well as the frequency of activated (CD69+) CD4 T cells in lymphoid organs, did not differ between mice with autophagy-competent and autophagy-deficient thymic epithelium. Inflammatory infiltrating cells, potentially indicative of autoimmune reactions, were present in the liver, lung, and colon of a similar fraction of ATG7 f/f and ATG7 f/f K14-Cre mice. In contrast to previously reported mice, that had received an autophagy-deficient thymus transplant, ATG7 f/f K14-Cre mice did not suffer from autoimmunity-induced weight loss. In summary, the results of this study suggest that autophagy in the thymic epithelium is dispensable for negative selection of autoreactive T cells.

Tissue-engineered calcium phosphate cement in rabbit femoral condylar bone defects

Background Calcium phosphate cement (CPC) is a favorable bone-graft substitute, with excellent biocompatibility and osteoconductivity. However, its reduced osteoinductive ability may limit the utility of CPC. To increase its osteoinductive potential, this study aimed to prepare tissue-engineered CPC and evaluate its use in the repair of bone defects. The fate of transplanted seed cells in vivo was observed at the same time. Methods Tissue-engineered CPC was prepared by seeding CPC with encapsulated bone mesenchymal stem cells (BMSCs) expressing recombinant human bone morphogenetic protein-2 (rhBMP-2) and green fluorescent protein (GFP). Tissue-engineered CPC and pure CPC were implanted into rabbit femoral condyle bone defects respectively. Twelve weeks later, radiographs, morphological observations, histomorphometrical evaluations, and in vivo tracing were performed. Results The radiographs revealed better absorption and faster new bone formation for tissue-engineered CPC than pure CPC. Morphological and histomorphometrical evaluations indicated that tissue-engineered CPC separated into numerous small blocks, with active absorption and reconstruction noted, whereas the residual CPC area was larger in the group treated with pure CPC. In the tissue-engineered CPC group, in vivo tracing revealed numerous cells expressing both GFP and rhBMP-2 that were distributed in the medullar cavity and on the surface of bony trabeculae. Conclusion Tissue-engineered CPC can effectively repair bone defects, with allogenic seeded cells able to grow and differentiate in vivo after transplantation.

Human enterovirus 71 (EV71) resisted type I interferon induced antiviral effect

To investigate human enterovirus 71 (EV71) resistance to type I interferon induced antiviral effect. After type I interferons (alpha, beta) were incubated with HeLa cells, recombinant type I herpes simple virus (HSV-1) with green fluorescent protein (GFP) was inoculated onto the HeLa cells. HSV-1 proliferation was observed by GFP expression and PCR. After EV71 was inoculated onto HeLa cells incubated with the same quantity of interferon, proliferation of EV71 were detected by RT-PCR of 2A gene. Recombinant HSV-1 GFP expression and viral DNA replication obviously decreased in HeLa cells incubated with type I interferon (alpha, beta). However, EV71 effectively proliferated in the interferon irritated HeLa cell by RT-PCR. HeLa cell irritated by type I interferon (alpha, beta) produced antiviral substance that inhibits HSV-1 proliferation. EV71 resisted the antiviral substance induced by type I interferon and could significantly replicate in the HeLa cells.

Retention of a recombinant GFP protein expressed by the yellow fever 17D virus in the E/NS1 intergenic region in the endoplasmic reticulum

The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein.

Expression of recombinant green fluorescent protein in Bacillus methanolicus

Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein.

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.

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4. The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein (1). The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH(4;))(2;)SO(4;)). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) (2). As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter (3). Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs (4). The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light (5). Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

Continuous-Flow Biomolecule and Cell Concentrator by Ion Concentration Polarization

We present a novel continuous-flow nanofluidic biomolecule/cell concentrator, utilizing the ion concentration polarization (ICP) phenomenon. The device has one main microchannel which bifurcates into two channels, one for a narrow, concentrated stream and the other for a wider but target-free stream. A nanojunction [cation-selective material (Nafion)] is patterned along the tilted concentrated channel. Application of an electric field generates the ICP zone near the nanojunction so that biomolecules and cells are guided into the narrow, concentrated channel by hydrodynamic force. Once biomolecules from the main channel are continuously streamed out to the concentrated channel, one can achieve a continuous flow of the same sample solution but with higher concentrations up to 100-fold. By controlling hydrodynamic resistance of the main and concentrated channel, the concentration factors can be adjusted. We demonstrated the continuous-flow concentration with various targets, such as bacteria [fluorescein sodium salt, recombinant green fluorescence protein (rGFP), red blood cells (RBCs), and Escherichia coli ( E. coli )]. Specially, fluorescein isothiocyanate (FITC)-conjugated lectin from Lens culinaris (lentil) (FITC-lectin) was tested on the different buffer conditions to clarify the effect of polarities of the target sample. This system is ideally suited for a generic concentration front-end for a wide variety of biosensors, with minimal integration-related complications.

Molecular designing for construction of a recombinant green fluorescent protein fused to single chain variable fragment of anti-human CD4 antibody

Molecular designing for construction of a recombinant green fluorescent protein fused to single chain variable fragment of anti-human CD4 antibody

Carbonaceous Materials Passivation on Amine Functionalized Magnetic Nanoparticles and Its Application for Metal Affinity Isolation of Recombinant Protein

Magnetic nanoparticles (MNPs) with an amine functionalized surface (MH) were passivated with carbonaceous materials (MH@C) by carbonization of glucose under hydrothermal reaction conditions. The carboxylate groups in carbonaceous shell could be enriched to 0.285 mmol/g when acrylic acid was added as a functional monomer in the carbonization reaction (MH@C-Ac). The carbonaceous shell not only protected the magnetic core from acidic erosion but also showed a high adsorption capacity toward Ni(2+) ion. The Ni(2+) ion complexed on MH@C and MH@C-Ac could specifically isolate 6×His tagged recombinant proteins from crude bacterial extracts via metal affinity interaction. The superparamagnetic property facilitates the easy retrieval of the carbonaceous material passivated MNPs from the viscous proteins solutions. Recombinant green fluorescence protein (GFP) and hyaluronic acid (HA) lyase of 9.4 mg and 2.3 mg could be isolated by 1 g of MH@C-Ac-Ni, respectively.

Simulation of dissolved CO2 gradients in a scale‐down system: A metabolic and transcriptional study of recombinant Escherichia coli

Deficient mixing in industrial-scale bioreactors is an important concern as it results in a heterogeneous environment that may affect microbial cell physiology. Dissolved carbon dioxide (dCO(2) ) fluctuations, which can occur in large-scale bioreactors, were simulated for the first time and their effects were evaluated on Escherichia coli expressing recombinant green fluorescent protein (GFP). The dCO(2) gradients were simulated by continuously circulating the medium between two vessels of a scale-down system to mimic mean circulation times (t(c) ) of 50, 170, and 375 s. Specific growth rate (μ) decreased by 11% and acetate concentration increased by 23% at the highest t(c) compared to reference cultures. An increase in the time needed for attaining maximum GFP concentration was also observed. The effect of dCO(2) fluctuations on the transcriptional levels of genes involved in the glutamate decarboxylase (gadA and gadC) and α-ketoglutarate dehydrogenase (sucA and sucB) were analyzed by quantitative real time PCR. Such genes are known to be highly over- or underexpressed at elevated constant dCO(2) . Expression levels of gadA and gadC increased up to 60% and 72%, and sucA decreased 1.8-fold in the culture performed at the highest t(c) . Only a minor decrease of sucB expression was observed at t(c) of 170 and 375 s. Although exposure to continuous high dCO(2) can affect culture performance, in this work it was shown that E. coli is able to rescue its metabolism in very short times when cells are intermittently returned to low dCO(2) conditions after being exposed to high dCO(2) .

Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

Immobilized metal ion affinity chromatography (IMAC) using peptide affinity tags has become a popular tool for protein purification. An important feature dictating the use of a specific affinity tag is whether its structure influences the properties of the target protein to which it is attached. In this work we have studied the influence on protein stability of two novel peptide affinity tags, namely NT1A and HIT2, and compared their effect to the commonly used hexa-histidine tag, all attached to the C-terminus of a enhanced green fluorescent protein (eGFP). A comparison of the influence of C- or N-terminal orientation of the tags was also carried out by studying the NT1A tag attached at either terminus of the eGFP. Protein stability was studied utilising guanidine hydrochloride equilibrium unfolding procedures and CD and fluorescence spectroscopy. The novel peptide affinity tags, NT1A and HIT2, and the His6 tag were found to not affect the stability of eGFP. Although these results are protein specific, they highlight, nevertheless, the need to employ suitable characterisation tools if the impact of a specific peptide tag on the folded status or stability of a recombinant tagged protein, purified by immobilized metal ion affinity chromatographic methods, are to be rigorously evaluated and the appropriate choice of peptide tag made.

Application of electrochemically produced aluminum hydroxide gel for prepurification of recombinant synthetic green fluorescent protein produced in tobacco leaves

The use of recombinant proteins has increased greatly in recent years, as also have increased the number of techniques and materials used for their production and purification. Among the different types of bioreactors being studied, there is a general consensus among scientists that production in green plant tissues such as leaves is more feasible. However, the presence of chlorophyll and phenolic compounds in plant extracts, which can precipitate and denature the proteins besides damaging separation membranes and gels, makes this technology impracticable on a commercial scale. In the present work, the adsorption to electrochemically produced aluminum hydroxide gel was applied as a prepurification step for recombinant synthetic green fluorescent protein (sGFP), also referred to as enhanced green fluorescent protein, produced in Nicotiana benthamiana leaves. Removal efficiencies of 99.7% of chlorophyll, 88.5% of phenolic compounds, and 38.5% of native proteins from the N. benthamiana extracts were achieved without removing sGFP from the extracts. As electrochemical preparation of aluminum hydroxide gel is a cost-effective technique, its use can substantially contribute to the development of future production platforms for recombinant proteins produced in green plant tissues of pharmaceutical and industrial interest.