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A cost effective process development for enhanced expression of antibody fragment in microbial cells, its impact on cellular physiology and transcriptome

ABSTRACT: A process for manufacturing the recombinant Ranibizumab has been developed with overall productivity of 0.72±0.02 mg/ml. Initially this study was attempted to find critical medium components based on literature. Among all the additives, glutamine, proline, arginine and ethanol showed increase in protein production compared to control. There was approximately 2 fold increase in Ranibizumab production in all the four additives analyzed using RP HPLC. Addition of glutamine, arginine and proline amino acids resulted in enhanced protein production however, the pH of the medium changed variably (pH 3.5-6.0). Since economic considerations decides the longevity of the process, as it plays a major role when large-scale operations are considered. Therefore, after economical potential analysis ethanol was finalized as the medium additive. Later, its effect was checked on cell size (control 1.82 µm and optimized 2µm), cellular physiology (no cell swelling or shrinkage was observed, cell width and length came out almost same in both the cases) and cellular viability (was found even better in optimized). Later, RNAseq was also performed to find out the underlying mechanism. Based on the literature as well as transcriptomic analysis and RT-qPCR validation, changes in membrane properties and DNA synthesis resulting in gene amplification that may have enhanced the synthesis of inducible proteins in case of optimized medium. The transcriptome studies always generates a huge dataset that can act as valuable resource for future studies to perform host cell engineering with the aim of improving heterologous protein expression.

ORGANISM(S): Escherichia coli

PROVIDER: GSE143359 | GEO | 2023/01/09

REPOSITORIES: GEO

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Evidence for proline utilisation by oral bacterial biofilms grown in saliva

Project description:Within the mouth bacteria are starved of saccharides as their main nutrient source between meals and it is unclear what drives their metabolism. Previously oral in vitro biofilms grown in saliva have shown proteolytic degradation of salivary proteins and increased extracellular proline. Although arginine and glucose have been shown before to have an effect on oral biofilm growth and activity, there is limited evidence for proline. Nuclear magnetic resonance (NMR) spectroscopy was used to identify extracellular metabolites produced by bacteria in oral biofilms grown on hydroxyapatite discs. Biofilms were inoculated with whole mouth saliva and then grown for 7 days using sterilised whole mouth saliva supplemented with proline, arginine and glucose as a growth-medium. Overall proline had a beneficial effect on biofilm growth – with significantly fewer dead bacteria present by biomass and surface area of the biofilms (p <0.05). Where arginine and glucose significantly increased and decreased pH, respectively, the pH of proline supplemented biofilms remained neutral at pH 7.3-7.5. SDS-polyacrylamide gel electrophoresis of the spent saliva from proline and arginine supplemented biofilms showed inhibition of salivary protein degradation of immature biofilms. NMR analysis of the spent saliva revealed that proline supplemented biofilms were metabolically similar to unsupplemented biofilms, but these biofilms actively metabolised proline to 5-aminopentanoate, butyrate and propionate, and actively utilised glycine. This study shows that in a nutrient limited environment, proline has a beneficial effect on in vitro oral biofilms grown from a saliva inoculum.

2020-07-23 | ST001433 | MetabolomicsWorkbench

Dynamic mRNA gene expression during a nutritional downshift from glutamine to proline

2015-04-07 | GSE54850 | GEO

Dynamic mRNA gene expression during a nutritional upshift from proline to glutamine

Project description:Dynamic mRNA gene expression from the wildtype YSBN6 during a nutritional upshift from proline to glutamine. Glutamine was added to yeast cells growing exponentially on proline as the sole nitrogen source.

2015-04-07 | GSE54844 | GEO

Dietary enrichments cause alterations in metabolome, transcriptome and improve freeze-tolerance in larvae of the fruit fly (Drosophila melanogaster).

Project description:In this experiment, Drosophila melanogaster larvae were fed with Proline and Arginine compounds and showed a better tolerance to freezing compared to control larvae. Using a microarray approach, we investigated the alteration of transcriptome profiles in larvae fed with Proline or Arginine versus control larvae.

2019-01-01 | E-MTAB-3911 | biostudies-arrayexpress

Expression data from Saccharomyces cerevisiae

Project description:Industrial bioethanol production may involve a low pH environment,improving the tolerance of S. cerevisiae to a low pH environment caused by inorganic acids may be of industrial importance to control bacterial contamination, increase ethanol yield and reduce production cost. Through analysis the transcriptomic data of Saccharomyces cerevisiae with different ploidy under low pH stress, we hope to find the tolerance mechanism of Saccharomyces cerevisiae to low pH.

2022-12-09 | GSE210964 | GEO

Combined Dietary and Pharmacological Depletion of Polyamines Targets Oncogenic Translation in Neuroblastoma

Project description:Cancer growth is fueled by nutrients obtained from circulation and local biosynthesis. Isolating and exploiting tumoral nutrient dependencies to potentiate current anti-cancer therapies is undergoing active research. We used unbiased metabolomics on patient samples and identified reprogramming of the arginine-proline-glutamine axis in MYCN amplified neuroblastoma. Tumoral acquisition of these non-essential amnio acids, as revealed by stable isotope tracing, was primarily by import, while extra-tumoral deamination of arginine indirectly feeds tumor ornithine via circulation. Dietary depletion of proline and arginine reduced systemic ornithine, the direct precursor of polyamine biosynthesis, and in combination with the clinically approved polyamine biosynthesis inhibitor, difluoromethylornithine, enhanced their depletion to improve survival in a MYCN-driven neuroblastoma mouse model. Mechanistically, ribosome profiling indicated specific translation defects, with ribosomal pausing at adenine-ending codons, to cause reprogramed protein biosynthesis affecting cell cycle and inducing neuronal differentiation. This work provides proof of concept for combined small molecule and nutrient depletion therapy for simultaneous targeting of translation in cancers dependent on external nutrient uptake.

2024-02-09 | GSE244378 | GEO

Optimization of SILAC in human embryonic stem cells

Project description:We present a fully defined culture system (adapted Essential8 (E8) medium in combination with vitronectin) for human embryonic stem cells (hESC) that can be used for Stable Isotopic Labeling of Amino aCids (SILAC) purposes. Although a complete incorporation of the labels was observed after 4 days in culture, more than 50 % of all mass spectrometry (MS) precursors displayed a conversion of L-arginine (R) to L-proline (P) or L-glumatate (E) of 40 %. To reduce this arginine conversion, E8 medium was modified by adding (1) L-proline, (2) L-ornithine, (3) Nω-hydroxy-nor-L-arginine (Nor-NOHA) acetate or by (4) lowering the arginine concentration. Inhibition of arginine conversion was best obtained by adding 5mM L-ornithine, followed by 3.5 mM L-proline and by lowering arginine concentration to 99.5 µM. No major changes in the proteome (HDMSE data), pluripotency and cell amount could be observed for these adapted Essential8TM media, although sudden cell death was sometimes observed with the use of 99.5 µM L-arginine. In conclusion, we suggest using 5 mM L-ornithine to reduce arginine conversion.

2016-07-15 | PXD002859 | Pride

Dynamic mRNA gene expression during a nutritional downshift from glutamine to proline

Project description:Dynamic mRNA gene expression from the wildtype YSBN6 during a nutritional downshift from glutamine to proline. Glutamine and proline were initially together in the media, with cells consuming exlusively glutamine (proline utilization inhibited due to nitrogen catabolite repression). The concentration of glutamine was frequently evaluated at-line, and the moment at which glutamine was not detected anymore is referred to as the time of the shift. Given the uncertainty of the exact time of the shift, two samples were taken at steady-state, approximately 10 and 5 minutes before the shift. Once the shift was identified, samples were taken 3, 7, 10, 14, 24, 56 and 120 minutes after the glutamine depletion. Biological triplicate gene expression was measured for samples -10, 7 and 24 minutes after shift, for a total of 15 chips. Changes were generally evaluated relative to the steady-state point (-10 minutes). Biological variability can be assessed from the replicates time points. Other dynamic omics data are associated with this dataset. Consult the publication for more details.

2015-04-07 | E-GEOD-54850 | biostudies-arrayexpress

p53 promotes cancer cell adaptation to glutamine deprivation by upregulating Slc7a3 to increase arginine uptake

Project description:Cancer cells heavily depend on the amino acid, glutamine, to meet the demands associated with growth and proliferation. Due to its rapid consumption, cancer cells frequently undergo glutamine starvation in vivo. We and others have shown that p53 is a critical regulator in metabolic stress resistance. To better understand the molecular mechanisms by which p53 activation promotes cancer cell adaptation to glutamine deprivation, we identified p53-dependent genes that are induced upon glutamine deprivation using RNA-seq analysis. We show that Slc7a3, an arginine transporter, is significantly induced by p53. We show the concomitant rise in intracellular arginine levels following glutamine deprivation is dependent on p53. The influx of arginine has minimal effect on known metabolic pathways upon glutamine deprivation. Instead, we found arginine serves as an effector for mTORC1 activation to promote in vitro and in vivo cell growth in response to glutamine starvation. Therefore, we identify a novel p53-inducible gene that contributes to the metabolic stress response.

2019-05-15 | GSE125782 | GEO

Chikungunya virus phosphoproteomics

Project description:SILAC-labeled MRC-5 cells were seeded in 75-cm2 flasks 1 day before infection with CHIKV-LS3 [1] at MOI 5. One hour post infection (h p.i.), the inoculum was removed and replaced with SILAC DMEM containing 2% dialyzed FBS, 0.280 mM arginine, 0.384 mM lysine, 0.5 mM proline, 25 mM HEPES, 2 mM L-Glutamine and 1% NEAA. At 2, 8, and 12 h p.i., infected and mock-infected cells were harvested for phosphoproteomics analysis by lysis in 4% SDS, 0.1M Tris pH 7.6, followed by heating to 96°C for 10 min. At 12 h p.i,. protein lysates for western blot (WB) analysis were harvested in 4× Laemmli sample buffer (LSB) (100 mM Tris-HCl, pH 6.8, 40% glycerol, 8% SDS, 40 mM DTT, 0,04 mg/ml bromophenol blue) and cells grown on coverslips were fixed in 3% PFA in PBS . The experiment was performed in duplicate with a label swap.

2022-02-28 | PXD009381 | Pride

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