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.
2024-08-13 | PXD042062 | Pride
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.
This model is hosted on
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and identified by:
MODEL1510220000.
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To the extent possible under law, all copyright and related or
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2024-10-15 | BIOMD0000001096 | BioModels
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:Recombinant Escherichia coli cultures are used to manufacture numerous therapeutic proteins and industrial enzymes, where many of these processes use elevated temperatures to induce recombinant protein production. The heat-shock response in wild-type E. coli has been well studied. In this study, the transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a dual heat-shock recombinant protein induction were examined. Most classical heat-shock protein genes were identified as regulated in both conditions. The major transcriptome differences between the recombinant and reported wild-type cultures were heavily populated by hypothetical and putative genes, which indicates recombinant cultures utilize many unique genes to respond to a heat-shock. Comparison of the dual stressed culture data with literature recombinant protein induced culture data revealed numerous differences. The dual stressed response encompassed three major response patterns: induced-like, in-between, and greater than either individual stress response. Also, there were no genes that only responded to the dual stress. The most interesting difference between the dual stressed and induced cultures was the amino acid-tRNA gene levels. The amino acid-tRNA genes were elevated for the dual cultures compared to the induced cultures. Since tRNAs facilitate protein synthesis via translation, this observed increase in amino acid-tRNA transcriptome levels, in concert with elevated heat-shock chaperones, might account for improved productivities often observed for thermo-inducible systems. Most importantly, the response of the recombinant cultures to a heat-shock was more profound than wild-type cultures, and further, the response to recombinant protein induction was not a simple additive response of the individual stresses. The objective of the present work is to gain a better understanding of the heat-shock response in recombinant cultures and how this response might impact recombinant protein production. To accomplish this objective, the transcriptome response of recombinant cultures subjected to a heat-shock and a dual heat-shock recombinant protein induction were analyzed. The transcriptome levels were determined using Affymetrix E. coli Antisense DNA microarrays, such that the entire genome was evaluated. These two transcriptome responses were also compared to recombinant cultures at normal growth temperature that were not over-expressing the recombinant protein and a set of literature recombinant culture data that were chemically induced to over-express the recombinant protein. Additionally, the heat-shock response of the recombinant cultures was compared to the literature report of the heat-shock response in wild-type cultures. The results of the global transcriptome analysis demonstrated that recombinant cultures respond differently to a heat-shock stress than wild-type cultures, where the transcriptome response of the recombinant cultures is further modified by production of a recombinant protein.
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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.
2020-12-21 | GSE108442 | GEO
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: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. EXPERIMENT: Mouse NS0 myeloma cells were grown in culture, stressed with 5 mM NaCl, 10 mM proline plus 5 mM ammonia, or 5 mM ammonia, along with an unstressed control group. The growth of the cultures were followed until the late exponential phase (90 hours), at which time two 50 mLs of cells were harvested and RNA extracted. Samples were prepared in triplicate, for a total of 12 samples. The RNA was amplified and labeled by Microarray Core (E) and hybridized to the GLYCOv3 microarrays.
2011-04-29 | E-GEOD-28920 | biostudies-arrayexpress