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Cell-Specific Labeling Enzymes For Analysis of Cell-Cell Communication in Continuous Co-Culture

ABSTRACT: We report the orthologous screening, engineering and optimization of amino acid conversion enzymes for cell-specific proteomic labeling.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christopher Tape

LAB HEAD: Claus Jorgensen

PROVIDER: PXD000739 | Pride | 2022-02-22

REPOSITORIES: pride

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Dataset's files

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			Action	DRS
	GFP_Day_0.pep.xml	Pepxml
	GFP_Day_0_A.raw	Raw
	GFP_Day_0_B.raw	Raw
	GFP_Day_0_C.raw	Raw
	GFP_Day_10_Continuous.pep.xml	Pepxml

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Publications

Cell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.

Tape Christopher J CJ Norrie Ida C IC Worboys Jonathan D JD Lim Lindsay L Lauffenburger Douglas A DA Jørgensen Claus C

Molecular & cellular proteomics : MCP 20140512 7

We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC(M.tub-KDEL)) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr(M37-KDEL)) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When e ...[more]

PMID: 24820872

Similar Datasets

Cell-Specific Labeling Enzymes For Analysis of Cell-Cell Communication in Continuous Co-Culture, Part 1 / 2

Project description:We report the orthologous screening, engineering and optimization of amino acid conversion enzymes for cell-specific proteomic labeling.

2014-05-29 | PXD000870 | Pride

Cell-Specific Labeling Enzymes For Analysis of Cell-Cell Communication in Continuous Co-Culture, Part 2 / 2

Project description:We report the orthologous screening, engineering and optimization of amino acid conversion enzymes for cell-specific proteomic labeling.

2014-05-29 | PXD000871 | Pride

Cell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.

Project description:We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC(M.tub-KDEL)) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr(M37-KDEL)) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDC(M.tub-KDEL) and Lyr(M37-KDEL) facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell-cell phospho-signaling experiments, we propose DDC(M.tub-KDEL) and Lyr(M37-KDEL) as excellent enzymes for cell-specific labeling with amino acid precursors.

| S-EPMC4083121 | biostudies-literature

Systematic evaluation of quantotypic peptides for targeted analysis of the human kinome

Project description:In targeted proteomics it is critical that peptides are not only proteotypic, but also accurately represent the level of the protein (quantotypic). Numerous approaches are used to identify proteotypic peptides, but quantotypic properties are rarely assessed. Here, we show that measuring ratios of proteotypic peptides across biological samples can be used to empirically identify peptides with good quantotypic properties, and use this to identify quantotypic peptides for 21% of the human kinome.

2014-08-08 | PXD001026 | Pride

Production of retroviral vectors in continuous high cell density culture.

Project description:Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting. KEY POINTS: • A continuous high cell density process for MLV vector production was established • The tubular cell retention membrane allowed for continuous vector harvesting • The established process had a 18-fold higher space time yield compared to a batch.

| S-EPMC10485120 | biostudies-literature

Continuous Live-Cell Culture Imaging and Single-Cell Tracking by Computational Lensfree LED Microscopy.

Project description:Continuous cell culture monitoring as a way of investigating growth, proliferation, and kinetics of biological experiments is in high demand. However, commercially available solutions are typically expensive and large in size. Digital inline-holographic microscopes (DIHM) can provide a cost-effective alternative to conventional microscopes, bridging the gap towards live-cell culture imaging. In this work, a DIHM is built from inexpensive components and applied to different cell cultures. The images are reconstructed by computational methods and the data are analyzed with particle detection and tracking methods. Counting of cells as well as movement tracking of living cells is demonstrated, showing the feasibility of using a field-portable DIHM for basic cell culture investigation and bringing about the potential to deeply understand cell motility.

| S-EPMC6427468 | biostudies-literature

Single-Use, Metabolite Absorbing, Resonant Transducer (SMART) Culture Vessels for Label-Free, Continuous Cell Culture Progression Monitoring.

Project description:Secreted metabolites are an important class of bio-process analytical technology (PAT) targets that can correlate to cell conditions. However, current strategies for measuring metabolites are limited to discrete measurements, resulting in limited understanding and ability for feedback control strategies. Herein, a continuous metabolite monitoring strategy is demonstrated using a single-use metabolite absorbing resonant transducer (SMART) to correlate with cell growth. Polyacrylate is shown to absorb secreted metabolites from living cells containing hydroxyl and alkenyl groups such as terpenoids, that act as a plasticizer. Upon softening, the polyacrylate irreversibly conformed into engineered voids above a resonant sensor, changing the local permittivity which is interrogated, contact-free, with a vector network analyzer. Compared to sensing using the intrinsic permittivity of cells, the SMART approach yields a 20-fold improvement in sensitivity. Tracking growth of many cell types such as Chinese hamster ovary, HEK293, K562, HeLa, and E. coli cells as well as perturbations in cell proliferation during drug screening assays are demonstrated. The sensor is benchmarked to show continuous measurement over six days, ability to track different growth conditions, selectivity to transducing active cell growth metabolites against other components found in the media, and feasibility to scale out for high throughput campaigns.

| S-EPMC11348071 | biostudies-literature

Chromatin state and chromatin architecture in mouse embryonic craniofacial tissue, neural crest cell culture model and human primary craniofacial tissue

Project description:Examination of the chromatin state and chromatin architecture of a region on human chromosome 7 and the orthologous region on mouse chromosome 6.

2023-03-30 | GSE198297 | GEO

Paired single cell co-culture microenvironments isolated by two-phase flow with continuous nutrient renewal.

Project description:Cancer-stromal cell interactions are a critical process in tumorigenesis. Conventional dish-based assays, which simply mix two cell types, have limitations in three aspects: 1) limited control of the cell microenvironment; 2) inability to study cell behavior in a single-cell manner; and 3) have difficulties in characterizing single cell behavior within a highly heterogeneous cell population (e.g. tumor). An innovative use of microfluidic technology is for improving the spatial resolution for single cell assays. However, it is challenging to isolate the paired interacting cells while maintaining nutrient renewal. In this work, two-phase flow was used as a simple isolation method, separating the microenvironment of each individual chamber. As nutrients in an isolated chamber are consumed by cells, media exchange is required. To connect the cell culture chamber to the media exchange layer, we demonstrated a 3D microsystem integration technique using vertical connections fabricated by deep reactive-ion etching (DRIE). Compared to previous approaches, the presented process allows area reduction of vertical connections by an order of magnitude, enabling compact 3D integration. A semi-permeable membrane was sandwiched between the cell culture layer and the media exchange layer. The selectivity of the semi-permeable membrane results in the retention of the signaling proteins within the chamber while allowing free diffusion of nutrients (e.g., glucose and amino acids). Thus, paracrine signals are accumulated inside the chamber without cross-talk between cells in other chambers. Utilizing these innovations, we co-cultured UM-SCC-1 (head and neck squamous cell carcinoma) cells and endothelial cells to simulate tumor proliferation enhancement in the vascular endothelial niche.

| S-EPMC4977363 | biostudies-literature

Continuous influenza virus production in cell culture shows a periodic accumulation of defective interfering particles.

Project description:Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. We explored the possibility to establish a continuous production process for influenza viruses using the duck-derived suspension cell line AGE1.CR. A two-stage bioreactor setup was designed in which cells were cultivated in a first stirred tank reactor where an almost constant cell concentration was maintained. Cells were then constantly fed to a second bioreactor where virus infection and replication took place. Using this two-stage reactor system, it was possible to continuously produce influenza viruses. Surprisingly, virus titers showed a periodic increase and decrease during the run-time of 17 days. These titer fluctuations were caused by the presence of defective interfering particles (DIPs), which we detected by PCR. Mathematical modeling confirmed this observation showing that constant virus titers can only emerge in the absence of DIPs. Even with very low amounts of DIPs in the seed virus and very low rates for de novo DIP generation, defective viruses rapidly accumulate and, therefore, represent a serious challenge for continuous vaccine production. Yet, the continuous replication of influenza virus using a two-stage bioreactor setup is a novel tool to study aspects of viral evolution and the impact of DIPs.

| S-EPMC3764112 | biostudies-literature

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