Genome-wide RNAi screen for improved functional expression of recombinant proteins from HEK 293 cells

For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. 21,585 human genes were individually silenced with three different siRNAs for each gene. 56 genes whose silencing caused the greatest improvement in the luciferase expression were found to be part of several different pathways that are associated with spliceosome formation/mRNA processing, transcription, metabolic process, transport and protein folding. 10 genes whose downregulation significantly enhanced the protein expression were validated by their silencing effect on four different recombinant proteins. Among the validated genes, the gene encoding the ornithine decarboxylase antizyme1- was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing this gene caused the increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine, and indicated that increased cellular polyamines enhanced luciferase expression without affecting its transcription. The study shows that this gene is a novel target for improving expression of recombinant proteins. The genome-scale screening demonstrated in this work can establish the foundation for targeted design of an efficient mammalian cell platform for different biotechnological applications

Glucose uptake regulation in E. coli by the small RNA SgrS: comparative analysis of E. coli K-12 (JM109 and MG1655) and E. coli B (BL21)

<p>Abstract</p> <p>Background</p> <p>The effect of high glucose concentration on the transcription levels of the small RNA SgrS and the messenger RNA ptsG, (encodin<it>g </it>the glucose transporter IICB^Glc), was studied in both <it>E. coli </it>K-12 (MG1655 and JM109) and <it>E. coli </it>B (BL21). It is known that the transcription level of <it>sgrS </it>increases when <it>E. coli </it>K-12 (MG1655 and JM109) is exposed to the non-metabolized glucose alpha methyl glucoside (αMG) or when the bacteria with a defective glycolysis pathway is grown in presence of glucose. The increased level of sRNA SgrS reduces the level of the ptsG mRNA and consequently lowers the level of the glucose transporter IICB^Glc. The suggested trigger for this action is the accumulation of the corresponding phospho-sugars.</p> <p>Results</p> <p>In the course of the described work, it was found that <it>E. coli </it>B (BL21) and <it>E. coli </it>K-12 (JM109 and MG1655) responded similarly to αMG: both strains increased <it>SgrS </it>transcription and reduced <it>ptsG </it>transcription. However, the two strains reacted differently to high glucose concentration (40 g/L). <it>E. coli </it>B (BL21) reacted by increasing <it>sgrS </it>transcription and reducing <it>ptsG </it>transcription while <it>E. coli </it>K-12 (JM109 and MG1655) did not respond to the high glucose concentration, and, therefore, transcription of <it>sgrS </it>was not detected and ptsG mRNA level was not affected.</p> <p>Conclusions</p> <p>The results suggest that <it>E. coli </it>B (BL21) tolerates high glucose concentration not only by its more efficient central carbon metabolism, but also by controlling the glucose transport into the cells regulated by the sRNA SgrS, which may suggest a way to control glucose consumption and increase its efficient utilization.</p

Expression of human α1-proteinase inhibitor in Aspergillus niger

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Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog

<p>Abstract</p> <p>Background</p> <p>Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.</p> <p>Results</p> <p>Two genes, cyclin-dependent kinase like 3 (<it>cdkl3</it>) and cytochrome c oxidase subunit (<it>cox15</it>), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of <it>cdkl3 </it>had a greater impact than that of <it>cox15</it>. Moreover, flow cytometric analysis indicated that cells with an insert of <it>cdkl3 </it>were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of <it>cox15 </it>was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than <it>cdkl3</it>. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either <it>cdkl3 </it>or <it>cox15</it>.</p> <p>Conclusion</p> <p>These results are consistent with previous studies on the functionalities of <it>cdkl3 </it>and <it>cox15</it>. For instance, the effect of <it>cdkl3 </it>on cell growth is consistent with its homology to the <it>cdk3 </it>gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to <it>cox15 </it>expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.</p

Improving production of retroviral vector from Pg13 cells for T cell therapy

Adoptive T-Cell therapy is a growing field for cancer treatment using the patient’s immune system to battle the cancer cells. Tumor specific T cells are either isolated from a tumor or created by modifying the T cells and after expansion are administered to the patient. The modifications include adding specific T cell receptors (TCR) or chimeric antigen receptors (CAR) by way of retroviral vector, lentiviral vector, or other method. One method is to use PG13 cells, which are derivatives of NIH3T3 mouse fibroblasts, to stably produce a retroviral vector that is used to transduce the T cell. PG13 cells are anchorage dependent cells that grow in roller bottles or cell factories to produce the viral vector and recently in a fixed bed bioreactor. To improve the production of the viral vector we explore the possibility of its production using PG13 cells grown on microcarriers in a bioreactor. Microcarriers are small, approximately 100-300 µm, charged beads that support the attachment of the cells and are suspended in the growth media in the bioreactor that provide controlled growth conditions. In this way parameters, such as oxygen concentration, pH, and nutrient are monitored and controlled. The result is higher cell concentration and consequently virus titer. There was no effect on the specific virus titer or the efficacy of the vector in transducing t cells indicating that using microcarriers in a bioreactor is a good method for scaling up stable production of gamma retroviral vector in PG13 cells

Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500

Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the expression approaches tested, introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is presently considered as a preferred method. In this presentation we will report on the effect of protective antigen expression on the metabolism of the producing train B. anthracis, BH500, by comparing it with a control strain carrying an empty plasmid. The two strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was performed. Several differences were observed, especially significant were the following: the strain expressing rPA showed increased transcription of sigL, the gene encoding RNA polymerase σ54, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. At the same time there were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Additionally, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain which was associated with decreased specific growth rates. The information and the observation acquired from this study will be presented together with possible approaches to create a better producing strain

Identifying Hipk1 as a target of Mir-22-3p enhancing recombinant protein production from Hek 293 by using microarray and Htp sirna screen

Enhancing protein production in mammalian cells is of interest in the biomedical field for a variety of reasons, including structural studies and antibody production. Using small non-protein coding RNA such as microRNA has recently been a promising method of increasing protein expression. A high throughput human microRNA screen in HEK 293 cells previously identified miRNA 22-3p as a promising candidate for increasing recombinant protein expression. This microRNA enhanced the expression of luciferase, two hard-to-express membrane proteins and a secreted hFc-fusion protein. In order to explore the mechanisms of this increase in protein production and to understand the intracellular events, we conducted a gene expression analysis of cells transfected with a mir-22-3p mimic against a negative control. Following the microarray analysis, several genes that were differentially regulated were identified. These were cross-referenced with predicted mir-22-3p targets along with the results of a high throughput siRNA screen. We will present our selected gene, HIPK1, and its possible involvement in the process of enhanced cells productivity

The role of Cra in regulating acetate excretion and osmotic tolerance in E. coli K-12 and E. coli B at high density growth

<p>Abstract</p> <p>Background</p> <p><it>E. coli </it>B (BL21), unlike <it>E.coli </it>K-12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate than <it>E. coli </it>K-12, especially when growing to high cell densities at high glucose concentration. By performing genomic analysis, it was demonstrated that the cause of this difference in sensitivity to the glucose concentration is the result of the differences in the central carbon metabolism activity. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in <it>E. coli </it>B and may be responsible for the different behaviour of the two strains. To investigate this possibility and better understand the function of Cra in the two strains, <it>cra </it>- negative <it>E. coli </it>B (BL21) and <it>E. coli </it>K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR.</p> <p>Results</p> <p>The deletion of the <it>cra </it>gene in <it>E. coli </it>B (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene in <it>E.coli </it>K-12 (JM109) caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. <it>ppsA </it>(gluconeogenesis gene), <it>aceBA </it>(the glyoxylate shunt genes) and <it>poxB </it>(the acetate producing gene) were down-regulated in both strains, while <it>acs </it>(acetate uptake gene) was down-regulated only in <it>E.coli </it>B (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth of <it>E. coli </it>K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing <it>bet </it>operon (<it>betABT</it>).</p> <p>Conclusions</p> <p>The transcriptional changes caused by the deletion of <it>cra </it>gene did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone; rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that <it>cra </it>deletion caused transcription inhibition of the <it>bet </it>operon in <it>E. coli </it>K-12 (JM109) but did not affect this operon transcription in <it>E. coli </it>B (BL21). This property, together with the insensitivity to high glucose concentrations, makes this the <it>E. coli </it>B (BL21) strain more resistant to environmental changes.</p

CHO microRNA engineering is growing up : recent successes and future challenges

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines

Bacillus subtilis contains multiple forms of somatostatin-like material

Extracts of B. subtilis contain somatostatin-like immunoactivity (1-20 pg per g wet weight cells). Two major forms were detected, one with reactivity in both N-and C- terminal immunoassays similar to somatostatin-28 and a second form reactive only in the C-terminal specific immunoassay similar to somatostatin-14. Both forms were active in a bioassay and the bioactivity was neutralized in the presence of antibody to the central, biologically active part of somatostatin-14. Preconditioned medium contained no detectable somatostatin whereas conditioned medium had 80-380 pg per liter.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25731/1/0000288.pd