Impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies of TEM1-beta-lactamase

The enzyme TEM1-beta-lactamase has been used as a model to study the impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies (IBs). The protein has been heterologously expressed in Escherichia coli in fed-batch cultivations at different temperatures (30, 37, and 40 degrees C) as well as induction regimes that guaranteed distinct product formation rates and ratios of soluble to aggregated protein. Additionally, shake flask cultivations at 20, 30, and 37 degrees C were performed. IBs were sampled during the whole bioprocess and structural analysis was performed by attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectroscopy. This work clearly demonstrates that the tested production regimes and rates had no impact on the IB structure, which was characterized by decreased alpha-helical and increased and modified beta-sheet contents compared to the native protein. Moreover, aggregates formed during refolding of IBs by solubilization and simple dilution showed very similar FT-IR spectra suggesting (i) the existence of only one critical folding step from which either aggregation (IB formation) or native folding branches off, and (ii) underlining the important role of the specific amino acid sequence in aggregation. The findings are discussed with respect to the known structure of TEM1-beta-lactamase and the reported kinetics of its (un)folding as well as contradictory data on the effect of cultivation regimes on IB structure(s) of other proteins.     

Disruption of hexokinase II (HXK2) partly relieves glucose repression to enhance production of human kringle fragment in gratuitous recombinant Saccharomyces cerevisiae

The GAL1 gene encoding galactokinase was disrupted in a recombinant Saccharomyces cerevisiae strain in which production of LK8 protein, a kringle fragment of human apolipoprotein, is under the control of GAL1 promoter. Null mutation of the HXK2 gene was introduced further in the gal1Delta strain to relieve glucose repression. A pattern for LK8 expression was compared for the two recombinant S. cerevisiae systems in continuous and fed-batch cultivations. A critical dilution rate in continuous cultivation that repressed LK8 expression was significantly higher for the gal1Deltahxk2Delta strain than that for the gal1Delta strain to sustain the LK8 production even at high glucose consumption rate. Expressed LK8 for the gal1Delta strain was not detectable when the dilution rate exceeded 0.05 h(-1). Maximum LK8 concentration of 57 mgl(-1) was obtained in glucose-limited fed-batch cultivation of the gal1Deltahxk2Delta strain, corresponding to a 13.8-fold enhancement compared with the gal1Delta strain grown under the same conditions.     

Wanted: more monitoring and control during inclusion body processing

Inclusion bodies (IBs) are insoluble aggregates of misfolded protein in Escherichia coli. Against the outdated belief that the production of IBs should be avoided during recombinant protein production, quite a number of recombinant products are currently produced as IBs, which are then processed to give correctly folded and soluble product. However, this processing is quite cumbersome comprising IB wash, IB solubilization and refolding. To date, IB processing often happens rather uncontrolled and relies on empiricism rather than sound process understanding. In this mini review we describe current efforts to introduce more monitoring and control in IB processes, focusing on the refolding step, and thus generate process understanding and knowledge.     

Intracellular aggregation of polypeptides with expanded polyglutamine domain is stimulated by stress-activated kinase MEKK1

Abnormal proteins, which escape chaperone-mediated refolding or proteasome-dependent degradation, aggregate and form inclusion bodies (IBs). In several neurodegenerative diseases, such IBs can be formed by proteins with expanded polyglutamine (polyQ) domains (e.g., huntingtin). This work studies the regulation of intracellular IB formation using an NH(2)-terminal fragment of huntingtin with expanded polyQ domain. We demonstrate that the active form of MEKK1, a protein kinase that regulates several stress-activated signaling cascades, stimulates formation of the IBs. This function of MEKK1 requires kinase activity, as the kinase-dead mutant of MEKK1 cannot stimulate this process. Exposure of cells to UV irradiation or cisplatin, both of which activate MEKK1, also augmented the formation of IBs. The polyQ-containing huntingtin fragment exists in cells in two distinct forms: (a) in a discrete soluble complex, and (b) in association with insoluble fraction. MEKK1 strongly stimulated recruitment of polyQ polypeptides into the particulate fraction. Notably, a large portion of the active form of MEKK1 was associated with the insoluble fraction, concentrating in discrete sites, and polyQ-containing IBs always colocalized with them. We suggest that MEKK1 is involved in a process of IB nucleation. MEKK1 also stimulated formation of IBs with two abnormal polypeptides lacking the polyQ domain, indicating that this kinase has a general effect on protein aggregation.     

Simulation of penicillin production in fed-batch cultivations using a morphologically structured model

A mathematical model is formulated to describe trends in biomass and penicillin formation as well as substrate consumption for fed-batch cultivations. The biomass is structured into three morphological compartments, and glucose and corn steep liquor are considered as substrates for growth. Penicillin formation is assumed to take place in the subapical compartment and in the growing region of the hyphal compartment. Furthermore, it is inhibited by glucose. Model parameters are estimated using an evolutionary algorithm and fitting the model to a standard fed-batch cultivation. The model is validated on experimental data from three different fed-batch cultivations, including two repeated fed-batch cultivations. The model predictions show good agreement with the measurements of biomass and pencillin concentrations for all fed-batch cultivations. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 593-604, 1997.     

Chemometric analysis of in-line multi-wavelength fluorescence measurements obtained during cultivations with a lipase producing Aspergillus oryzae strain

The filamentous fungus, Aspergillus oryzae, was cultivated in batch and fed-batch cultivations in order to investigate the use of multi-wavelength fluorescence for monitoring course of events during filamentous fungi cultivations. The A. oryzae strain applied expressed a fungal lipase from Thermomyces lanuginosus. Spectra of multi-wavelength fluorescence were collected every 5 min with the BioView system (DELTA, Denmark) and both explorative and predictive models, correlating the fluorescence data with cell mass and lipase activity, were built. During the cultivations, A. oryzae displayed dispersed hyphal growth and under these conditions no fouling of the multi-wavelength fluorescence sensor was observed. The scores of a parallel factor analysis (PARAFAC) model, based on the fluorescence spectra, gave clear evidence of, for example, the on-set of the feeding phase. The predictive models, estimating the cell mass, showed correlations between 0.73 and 0.97 with root mean square error of cross validation (RMSECV) values between 1.48 and 0.77 g . kg(-1). A model estimating the lipase activity was also constructed for the fed-batch cultivations with a correlation of 0.93. The results presented here clearly show that multi-wavelength fluorescence is a useful tool for monitoring fed-batch cultivations of filamentous fungi.     

On-line monitoring of recombinant bacterial cultures using multi-wavelength fluorescence spectroscopy

Multi-wavelength fluorescence spectroscopy was evaluated as a tool for on-line monitoring of recombinant Escherichia coli cultivations expressing human basic fibroblast growth factor (hFGF-2). The data sets for the various combinations of the excitation and emission spectra from batch cultivations were analyzed using principal component analysis. Chemometric models (the partial least squares method) were developed for correlating the fluorescence data and the experimentally measured variables such as the biomass and glucose concentrations as well as the carbon dioxide production rate. Excellent correlations were obtained for these variables for the calibration cultivations. The predictability of these models was further tested in batch and fed-batch cultivations. The batch cultivations were well predicted by the PLS models for biomass, glucose concentrations and carbon dioxide production rate (RMSEPs were respectively 5%, 7%, 9%). However, when tested for biomass concentrations in fed-batch cultivations (with final biomass three times higher than the highest calibration data) the models had good predictability at high growth rates (RMSEPs were 3% and 4%, respectively for uninduced and induced fed-batch cultivations), which was as good as for the batch cultivations used for developing the models (RMSEPs were 3% and 5%, respectively for uninduced and induced batch cultivations). The fed-batch cultivations performed at low growth rates exhibited much higher fluorescence for fluorophores such as flavin and NAD(P)H as compared to fed-batch cultivations at high growth rate. Therefore, the PLS models tended to over-predict the biomass concentrations at low growth rates. Obviously the cells changed their concentration of biogenic fluorophores depending on the growth rate. Although multi-wavelength fluorescence spectroscopy is a valuable tool for on-line monitoring of bioprocess, care must be taken to re-calibrate the PLS models at different growth rates to improve the accuracy of predictions.     

Calorimetric control of fed-batch cultures of Saccharomyces cerevisiae

Calorimetry has been used to control the glucose feeding in fed-batch cultures of S. cerevisiae in order to avoid ethanol formation and maintain a fully respiratory metabolism. Comparisons between batch and fed-batch cultivations showed that the former had a much lower growth yield. The growth yields for fed-batch cultivations were more than 30% higher than for batch cultures. However, energy balance calculations showed that a large part of the increase could be explained by the evaporation of ethanol during batch cultivations. When the growth yields obtained from the batch cultures were corrected for the evaporation of ethanol, the increase in growth yield for fed-batch cultures was about 10%.     

Reduction of N-terminal methionylation while increasing titer by lowering metabolic and protein production rates in E. coli auto-induced fed-batch fermentation

A standard fed-batch fermentation process using 1 mM isopropyl-β-D: -thiogalactopyranoside (IPTG) induction at 37 °C in complex batch and feed media had been developed for manufacturing of a therapeutic protein (TP) expressed in inclusion bodies (IBs) by E. coli BL21 (DE3) driven by T7 promoter. Six unauthentic TP N-terminal variants were identified, of which methionylated TP (Met-TP) ratio was predominant. We hypothesized that lowering metabolic and protein production rates would reduce the Met-TP ratio while improving TP titer. The standard process was surprisingly auto-induced without added IPTG due to galactose in the complex media. Without changing either the clone or the batch medium, a new process was developed using lower feed rates and auto-induction at 29 °C after glucose depletion while increasing induction duration. In comparison to the standard process, the new process reduced the unauthentic Met-TP ratio from 23.6 to 9.6 %, increased the TP titer by 85 %, and the specific production yield from 210 to 330 mg TP per gram of dry cell weight. Furthermore, the TP recovery yield in the purified IBs was improved by ~20 %. Adding together, ~105 % more TP recovered in the purified IBs from per liter of fermentation broth for the new process than the standard process. The basic principles of lowering metabolic and production rates should be applicable to other recombinant protein production in IBs by fed-batch fermentations.     

High throughput inclusion body sizing: Nano particle tracking analysis

The expression of pharmaceutical relevant proteins in Escherichia coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In the scientific literature, substantial effort has been devoted to the quantification of IB size. However, particle‐based methods used up to this point to analyze the physical properties of representative numbers of IBs lack sensitivity and/or orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells. TEM imaging in combination with manual grey scale image segmentation allowed the quantification of relative areas covered by the inclusion body within the cytosol. As a high throughput method nano particle tracking analysis (NTA) enables one to derive the diameter of inclusion bodies in cell homogenate based on a measurement of the Brownian motion. The NTA analysis of fixated (glutaraldehyde) and non‐fixated IBs suggests that high pressure homogenization annihilates the native physiological shape of IBs. Nevertheless, the ratio of particle counts of non‐fixated and fixated samples could potentially serve as factor for particle stickiness. In this contribution, we establish image segmentation of TEM pictures as an orthogonal method to size biologic particles in the cytosol of cells. More importantly, NTA has been established as a particle‐based, fast and high throughput method (1000–3000 particles), thus constituting a much more accurate and representative analysis than currently available methods.     

Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli

A simple pulse-based method for the determination of the maximum uptake capacities for glucose and oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where specific growth rate was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when specific growth rate declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.     

Fibroblast growth factor homologous factors and the islet brain-2 scaffold protein regulate activation of a stress-activated protein kinase

Fibroblast growth factor homologous factors (FHFs) form native intracellular complexes with the mitogen-activated protein kinase (MAPK) scaffold protein islet-brain 2 (IB2) in adult brain. FHF binding to IB2 facilitates recruitment of the MAPK p38delta (SAPK4), while failing to stimulate binding of JNK, the preferred kinase of the related scaffold IB1 (JIP-1). We now report further biochemical evidence supporting FHFs as regulators of IB2 scaffold activity. Mixed lineage kinase 3 (MLK3) and IB2 synergistically activate p38delta but not the MAPKs JNK-1 and p38alpha. Binding of p38delta to IB2 is mediated by the carboxyl-terminal half of the scaffold (IB2(Delta1-436)). FHF2 also binds weakly to IB2(Delta1-436) and can thereby increase p38delta interaction with IB2(Delta1-436). FHF-induced recruitment of p38delta to IB2 is accompanied by increased levels of activated p38delta, and synergistic activation of p38delta by MLK3 and IB2 is further enhanced by FHF2. Consistent with a role for FHFs as signaling molecules, FHF2 isolated from rat brain is serine/threonine-phosphorylated, and FHF can serve as a substrate for p38delta in vitro. These results support the existence of a signaling module in which IB2 scaffolds a MLK3/MKK/p38delta kinase cascade. FHFs aid in recruitment of p38 to IB2 and may serve as kinase substrates.     

The economics of inclusion body processing

Many recombinant proteins are often over-expressed in host cells, such as Escherichia coli, and are found as insoluble and inactive protein aggregates known as inclusion bodies (IBs). Recently, a novel process for IB extraction and solubilisation, based on chemical extraction, has been reported. While this method has the potential to radically intensify traditional IB processing, the process economics of the new technique have yet to be reported. This study focuses on the evaluation of process economics for several IB processing schemes based on chemical extraction and/or traditional techniques. Simulations and economic analysis were conducted at various processing conditions using granulocyte macrophage-colony stimulating factor, expressed as IBs in E. coli, as a model protein. In most cases, IB processing schemes based on chemical extraction having a shorter downstream cascade demonstrated a competitive economic edge over the conventional route, validating the new process as an economically more viable alternative for IB processing.     

Ataxin-1 fusion partners alter polyQ lethality and aggregation

Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8+/-4.5% cell death versus 12.85+/-3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality.     

Packaging protein drugs as bacterial inclusion bodies for therapeutic applications

ABSTRACT: A growing number of insights on the biology of bacterial inclusion bodies (IBs) have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70), enzymes (catalase and dihydrofolate reductase), grow factors (leukemia inhibitory factor, LIF) and structural proteins (the cytoskeleton keratin 14) have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus) empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills), for their extra- or intra-cellular release in medicine and cosmetics.     

Single Neuron Ubiquitin-Proteasome Dynamics Accompanying Inclusion Body Formation in Huntington Disease

The accumulation of mutant protein in intracellular aggregates is a common feature of neurodegenerative disease. In Huntington disease, mutant huntingtin leads to inclusion body (IB) formation and neuronal toxicity. Impairment of the ubiquitin-proteasome system (UPS) has been implicated in IB formation and Huntington disease pathogenesis. However, IBs form asynchronously in only a subset of cells with mutant huntingtin, and the relationship between IB formation and UPS function has been difficult to elucidate. Here, we applied single-cell longitudinal acquisition and analysis to monitor mutant huntingtin IB formation, UPS function, and neuronal toxicity. We found that proteasome inhibition is toxic to striatal neurons in a dose-dependent fashion. Before IB formation, the UPS is more impaired in neurons that go on to form IBs than in those that do not. After forming IBs, impairment is lower in neurons with IBs than in those without. These findings suggest IBs are a protective cellular response to mutant protein mediated in part by improving intracellular protein degradation.Huntington disease (HD)⁴ is a progressive incurable neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) stretch in the N-terminal end of the huntingtin (htt) protein above a threshold length of ∼36 (1). The deposition of polyQ-expanded aggregated mutant htt in inclusion bodies (IBs) is a hallmark of HD, and IBs are found in human post-mortem samples, transgenic mouse brain, and cell-culture models (2). The accumulation of ubiquitinated proteins in IBs has implicated the ubiquitin-proteasome system (UPS) in the pathogenesis of HD, amyotrophic lateral sclerosis, Parkinson disease, and polyQ-mediated disorders (3).The UPS is a major pathway of intracellular protein degradation. After a series of three reactions, each catalyzed by a different set of enzymes, ubiquitin, a 76-amino acid polypeptide, forms an isopeptide bond with the amino group of lysine residues on substrate proteins. Several lysine residues within ubiquitin are sites for more ubiquitin additions. Once a protein accumulates four or more ubiquitins, it is efficiently targeted to the proteasome for degradation. The proteasome binds polyubiquitinated substrates and hydrolyzes ubiquitin isopeptide bonds, releasing ubiquitin moieties before degrading substrate proteins through chymotrypsin-like, trypsin-like, and post-glutamyl peptidase activities (3).Increased polyubiquitin levels and changes in ubiquitin linkages accompany the accumulation of UPS substrates in the brains of HD patients and transgenic mice and in cellular HD models (4). UPS substrates accumulate throughout the cell in polyQ models, even before IB formation (5, 6). This has added to the confusion over whether polyQ expansion leads to toxicity through direct impairment of proteasomal degradation. Proteasomes have been reported to cleave polyQ stretches efficiently (7), inefficiently (8), or essentially not at all (9). In vivo, polyQ-dependent degeneration occurs with no detectable proteasome inhibition (10, 11) or is tightly linked to it (12, 13). The inability of some studies to detect UPS impairment in HD models may be due to the limited sensitivity of conventional approaches to identify cell-to-cell variations in UPS function.The relationship between IB formation and UPS function has been difficult to determine. Protein turnover in cells with IBs is evidently reduced and accompanied by the accumulation of cellular proteins (14–16); HEK293 cells containing mutant htt IBs have a greater degree of UPS impairment than those without IBs (5). Proteasome subunits and heat shock proteins colocalize with IBs, but it is unclear if this colocalization facilitates protein delivery or unfolding at the mouth of active proteasomes, or if it harms proteasome function by sequestering essential cellular machinery (18). Some IBs are relatively static (8, 25), but the proteins in others are dynamically exchanged with cytoplasmic and nuclear pools (19, 20).UPS function is critical to cellular homeostasis. Deletion of one of the two inducible polyubiquitin genes in mice leads to lower intracellular ubiquitin levels in germ cells and hypothalamic neurons. These same populations undergo cell-cycle arrest and hypothalamic neurodegeneration, respectively (22, 23). Cell lines expressing mutant huntingtin accumulate ubiquitinated proteins and undergo cell-cycle arrest in G2/M (5). In neurons, UPS impairment may lead to cell death through an accumulation of signals for apoptosis, a decrease in NF-κB signaling, sensitization to other toxic stimuli, remodeling of synapses, retraction of neurites, or other unidentified mechanisms (24). The effect of UPS impairment depends on cell type and cell cycle, and the relationship between UPS impairment and striatal neuronal survival is largely unknown.Diffuse species of mutant htt induce IB formation and neuronal death in a protein concentration-dependent manner (2). IB formation delays neuronal death, suggesting that IB formation helps neurons cope with toxic diffuse mutant htt. Whether the effect of IB formation on survival is mediated through UPS function has been difficult to determine. IB formation and neuronal death occur asynchronously in overlapping but distinct subsets of neurons that express mutant htt. The observation that IB formation is not required for UPS impairment also complicates population analysis (6, 26).To explore this problem, we applied single-cell analysis. We tracked single neurons over their entire lifetimes, gaining spatial and temporal resolution while simultaneously monitoring IB formation, UPS inhibition, and neuronal toxicity.     

Intracellular pH-based controlled cultivation of yeast cells: II. cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. (c) 1993 John Wiley & Sons, Inc.     

Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS

Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.     

Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His₆ tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L⁻¹ could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.     

唇?硬骨蛋白基因克隆及其在肌间刺发生过程中的表达

为探究硬化蛋白(Sclerostin, SOST)与肌间刺发生之间的关系, 研究通过转录组测序和RT-PCR获得了唇?(Hemibarbus labeo)硬化蛋白基因cDNA序列。序列分析表明, 硬化蛋白由信号肽和成熟肽两部分组成, 成熟肽包含一个胱氨酸结样结构域。系统进化树分析表明, 唇?硬化蛋白与金鱼(Carassius auratus)硬化蛋白最为接近。通过荧光定量PCR检测发现唇?硬化蛋白基因mRNA在所有被检测的组织中均有表达, 在鳃中的表达量最高。RNA原位杂交结果表明, 硬化蛋白mRNA分布于肌膈, 且随着肌间刺的发生信号逐步减弱。RT-qPCR显示, 在肌间刺4个发育阶段中, 硬化蛋白基因mRNA的变化显著, 在阶段Ⅰ表达量最高, 随后逐步降低。综上, 唇?硬化蛋白与肌间刺骨化存在一定的相关性。研究结果将为进一步调查鱼类硬化蛋白功能及硬化蛋白在肌间刺形成过程中的作用提供理论依据。