Recombinant Cypridina luciferase
Ontology highlight
ABSTRACT:
ORGANISM(S): Cellular Organisms
SUBMITTER: Hiroyuki Kaji
PROVIDER: PXD029916 | JPOST Repository | Fri Nov 25 00:00:00 GMT 2022
REPOSITORIES: jPOST
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Project description:Recombinant TLP (rTLP) and CHI (rCHI), expressed by Komagataella phaffii, were used as as haze-protein models, for having similar characteristics (aggregation potential, melting point, functionality, glycosylation levels and bentonite adsorption) to the native-haze proteins from Vitis vinifera.
2023-03-11 | PXD035796 | Pride
Project description:Elevated N-linked glycosylation of immunoglobulin G variable regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design included adaptive immune receptor repertoire sequencing to quantify and characterize N-glycosylation sites in the global B cell receptor repertoire, proteomics to examine glycosylation patterns of the circulating IgG, and production of human-derived recombinant autoantibodies, which were studied with mass spectrometry and antigen binding assays to confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the B cell repertoire of MG patients when compared to healthy donors. Motifs were introduced by both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the circulating IgG in a subset of MG patients. Autoantigen binding, by patient-derived MG autoantigen-specific monoclonal antibodies with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of variable region N-linked glycosylation in autoimmunity to MG subtypes. Although occupied IgG-VN-Glyc motifs are found on MG autoantigen-specific monoclonal antibodies, they are not required for binding to the autoantigen in this disease.
2021-08-13 | GSE182038 | GEO
Project description:Recombinant proteins are of great interest in glycobiology and proteomics, known especially for their reproducibility and accessibility. However, variation in glycosylation among recombinant glycoproteins is not well understood and may depend on numerous conditions in the biomanufacturing process. In order to confidently assess variation in glycosylation measurements, it is vital to both optimize the measurement of, and determine the degree of variation between, distributions of glycosylation on specific sites of glycoproteins. This is especially important for glycoproteins that are known to have rapid sequence changes, such as with different influenza strains. In this study, eight strains of recombinant influenza hemagglutinin and neuraminidase produced from HEK293 cell line were obtained from four vendors and digestion was conducted using a series of complex multi-enzymatic methods designed to isolate glycopeptide sequons. Site-specific glycosylation profiles of intact glycopeptides were produced using mass spectrometric evaluation on an orbitrap system and compared using spectral similarity scores. Variation in glycan abundances and distribution was most pronounced between different strains of virus (similarity score = 383 out of 1000), whereas replicates resulted in low variation (similarity score = 957 out of 1000). Glycan variation was also measured based on differences between vendors, lots, batches, protease digestion, and intra-protein site. The most abundant glycans in all of these influenza glycoproteins were monofucosylated and complex, as reported by other laboratories. However, it was found that different vendors can produce very different glycan distributions for the same glycosylation site. Notably, it is demonstrated that glycan distributions are similar for conserved regions of influenza glycoproteins. Overall, these methods present a potential use in developing reproducible measurements of glycosylated biologics for quality control or making more informed decisions in biomanufacturing.
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Project description:Irani2015 - Genome-scale metabolic model of
P.pastoris N-glycosylation
This model is described in the article:
Genome-scale metabolic model
of Pichia pastoris with native and humanized glycosylation of
recombinant proteins.
Irani ZA, Kerkhoven E, Shojaosadati
SA, Nielsen J.
Biotechnol. Bioeng. 2015 Oct;
Abstract:
Pichia pastoris is used for commercial production of human
therapeutic proteins, and genome-scale models of P. pastoris
metabolism have been generated in the past to study the
metabolism and associated protein production by this yeast. A
major challenge with clinical usage of recombinant proteins
produced by P. pastoris is the difference in N-glycosylation of
proteins produced by humans and this yeast. However, through
metabolic engineering a P. pastoris strain capable of producing
humanized N-glycosylated proteins was constructed. The current
genome-scale models of P. pastoris do not address native nor
humanized N-glycosylation, and we therefore developed
ihGlycopastoris, an extension to the iLC915 model with both
native and humanized N-glycosylation for recombinant protein
production, but also an estimation of N-glycosylation of P.
pastoris native proteins. This new model gives a better
predictions of protein yield, demonstrates the effect of the
different types of N-glycosylation of protein yield, and can be
used to predict potential targets for strain improvement. The
model represents a step towards a more complete description of
protein production in P. pastoris, which is required for using
these models to understand and optimize protein production
processes. This article is protected by copyright. All rights
reserved.
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2024-10-15 | BIOMD0000001096 | BioModels
Project description:In this study, we focused on understanding the effect of the functional inactivation of the caspase 8 associated protein 2 (CASP8AP2) gene on gene expression in HEK293 cells, to possibly explain the improvement in recombinant protein expression engendered by its inactivation. To achieve this goal, the HEK293 cell line constitutively expressing luciferase was treated with CRISPR constructs targeting the CASP8AP2 gene, single clones isolated and a clone with a functionally compromised CASP8AP2 gene, 892-7, selected for further investigations. Recombinant luciferase expression, recombinant secreted alkaline phosphatase expression, growth and metabolic characteristics were evaluated and compared between the 892-7 clone and the parental cell line. Cells cultured to a confluence of about 80% were used for gene expression profiling. We found that genes involved in cell cycle progression and in histone mRNA synthesis were highly differentially expressed between the two cell lines.
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Project description:These research areas concentrate on stress induced proteases in recombinant Escherichia coli, glycosylation heterogeneity due to bioprocess conditions produced in mammalian cells, and metabolic engineering of E. coli. The hypothesis of this project is that recombinant protein glycosylation is inefficient under normal bioreactor conditions since the additional glycosylation reactions necessary for the recombinant protein exceed the metabolic capacity of the cells. Normal bioreactor conditions have been optimized for cell growth, and sometimes for protein productivity. Only recently has it been accepted that optimal glycosylation may not occur under optimal growth or protein productivity conditions. Specific Aim: Determine the relationship between bioreactor conditions and glycosylation gene expression in NS0 cells.
2011-04-29 | GSE28920 | GEO
Project description:Overall aim of this study was to investigate the role of up-regulated genes in the generation of cellular stress response that is triggered upon induction of recombinant protein synthesis. Up-regulated genes identified previously by transcriptomic analysis, were knocked out from the host genome and their impact on cellular health and expression capabilities was analyzed. Transcriptomic profiling of the top performing double knock out (∆(elaA+cysW) having significantly higher protein expression levels was compared with the control to demonstrate the ability of this knock-out strain to counter cellular stress.
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Project description:In order to explore how S100A8 and S100A9 may participate in the kidney stone formation, we used recombinant S100A8, recombinant S100A9, or recombinant S100A8/S100A9 heterodimer to culture the HK-2 cells and then sequenced total cellular mRNAS.
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Project description:The Schlafen (SLFN) family of nucleic acid binding proteins play an important role in gene regulation. In response to environmental stress, many SLFN members are activated for RNA cleavage and translational regulation. To characterize the RNA cleavage activity of this protein family, we produced recombinant human SLFN11 and SLFN14 protein. We utilized mass spectrometry for protein identification to confirm the identity of our recombinant SLFN protein samples.
2025-07-08 | PXD064237 | Pride
Project description:In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein in Escherichia coli cells. This metabolic burden analysis was performed against a number of recombinant expression systems and control strains and included: (i) standard transcriptional recombinant expression control system BL21(DE3) with the expression plasmid pET-eGFP, (ii) the recently developed dual transcriptional–translational recombinant expression control strain BL21(IL3), with pET-eGFP, (iii) BL21(DE3) with an empty expression plasmid pET, (iv) BL21(IL3) with an empty expression plasmid, and (v) BL21(DE3) without an expression plasmid; all strains were cultured under various induction conditions. The growth profiles of all strains together with the results gathered by the analysis of the Fourier transform infrared (FT-IR) spectroscopy data, identified IPTG-dependent induction as the dominant factor hampering cellular growth and metabolism, which was in general agreement with the findings of GC-MS analysis of cell extracts and media samples. In addition, the exposure of host cells to the synthetic inducer ligand, pyrimido[4,5-d] pyrimidine-2,4-diamine (PPDA), of the orthogonal riboswitch containing expression system (BL21(IL3)) did not display any detrimental effects, and its detected levels in all the samples were at similar levels, emphasising the inability of the cells to metabolise PPDA. The overall results obtained in this study suggested that although the BL21(DE3)-EGFP and BL21(IL3)-EGFP strains produced comparable levels of recombinant eGFP, the presence of the orthogonal riboswitch seemed to be moderating the metabolic burden of eGFP production in the cells enabling higher biomass yield, whilst providing a greater level of control over protein expression.
2017-11-16 | MTBLS393 | MetaboLights