Targeted genetic modification of cell lines for recombinant protein production.
Ontology highlight
ABSTRACT:
SUBMITTER: Barron N
PROVIDER: S-EPMC2267619 | biostudies-other | 2007 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Declining fossil fuel reserves, coupled with environmental concerns over their continued extraction and exploitation have led to strenuous efforts to identify renewable routes to energy and fuels. One attractive option is to convert glycerol, a by-product of the biodiesel industry, into n-butanol, an industrially important chemical and potential liquid transportation fuel, using Clostridium pasteurianum. Under certain growth conditions this Clostridium species has been shown to predominantly produce n-butanol, together with ethanol and 1,3-propanediol, when grown on glycerol. Further increases in the yields of n-butanol produced by C. pasteurianum could be accomplished through rational metabolic engineering of the strain. Accordingly, in the current report we have developed and exemplified a robust tool kit for the metabolic engineering of C. pasteurianum and used the system to make the first reported in-frame deletion mutants of pivotal genes involved in solvent production, namely hydA (hydrogenase), rex (Redox response regulator) and dhaBCE (glycerol dehydratase). We were, for the first time in C. pasteurianum, able to eliminate 1,3-propanediol synthesis and demonstrate its production was essential for growth on glycerol as a carbon source. Inactivation of both rex and hydA resulted in increased n-butanol titres, representing the first steps towards improving the utilisation of C. pasteurianum as a chassis for the industrial production of this important chemical.
| S-EPMC5367854 | biostudies-literature
Project description:Genetically encoded unnatural amino acids provide powerful strategies for modulating the molecular functions of proteins in mammalian cells. However, this approach has not been coupled to genome-wide measurements, because efficient incorporation of unnatural amino acids is limited to transient expression settings that lead to very heterogeneous expression. We demonstrate that stable integration of the Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)/tRNA(Pyl)CUA pair (and its derivatives) into the mammalian genome enables efficient, homogeneous incorporation of unnatural amino acids into target proteins in diverse mammalian cells, and we reveal the distinct transcriptional responses of embryonic stem cells and mouse embryonic fibroblasts to amber codon suppression. Genetically encoding N-?-acetyl-lysine in place of six lysine residues in histone H3 enables deposition of pre-acetylated histones into cellular chromatin, via a pathway that is orthogonal to enzymatic modification. After synthetically encoding lysine-acetylation at natural modification sites, we determined the consequences of acetylation at specific amino acids in histones for gene expression.
| S-EPMC4888942 | biostudies-literature
Project description:During the development of cell lines for therapeutic protein production, a vector harboring a product transgene is integrated into the genome. To ensure production stability and consistent product quality, single-cell cloning is then performed. Since cells derived from the same parental clone have the same transgene integration locus, the identity of the integration site can also be used to verify the clonality of a production cell line. In this study, we present a high-throughput pipeline for clonality verification through integration site analysis. Sequence capture of genomic fragments that contain both vector and host cell genome sequences was used followed by next-generation sequencing to sequence the relevant vector-genome junctions. A Python algorithm was then developed for integration site identification and validated using a cell line with known integration sites. Using this system, we identified the integration sites of the host vector for 31 clonal cell lines from five independent vector integration events while using one set of probes against common features of the host vector for transgene integration. Cell lines from the same lineage had common integration sites, and they were distinct from unrelated cell lines. The integration sites obtained for each clone as part of the analysis may also be used for clone selection, as the sites can have a profound effect on the transgene's transcript level and the stability of the resulting cell line. This method thus provides a rapid system for integration site identification and clonality verification.
| S-EPMC7803388 | biostudies-literature
Project description:Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signalling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure-function relationships, kinase signalling networks and kinase inhibitor drugs.
| S-EPMC4566161 | biostudies-literature
Project description:The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1. In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the Deltacre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T. reesei.
| S-EPMC2708423 | biostudies-literature
Project description:Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.
2020-03-19 | GSE144624 | GEO
Project description:Direct oral anticoagulants (DOAC) are substrates for the ABCB1 transporter (also called P-glycoprotein), an active efflux pump. ABCB1 polymorphisms have been previously reported to influence the pharmacokinetics of several drugs such as immunosuppressants and tyrosine kinase inhibitors. Recently, in vivo studies have suggested that genetic variants might contribute to the inter-individual variability in DOAC plasma concentrations. Therefore, we evaluated the in vitro effect of the most common coding ABCB1 single nucleotide polymorphisms (SNP), 1236?C?>?T-2677G?>?T-3435C?>?T, and the coding ABCB1 1199?G?>?A SNP on the transport activity towards rivaroxaban. HEK293 cells were transfected to overexpress the ABCB1 wild-type (1236C-2677G-3435C, 1199?G) or variant proteins (1236C-2677G-3435T, 1236T-2677T-3435T or 1199?A). ABCB1 expression decreased the intracellular accumulation of rivaroxaban, when compared to control cells. This confirms the involvement of ABCB1 in the active transport of rivaroxaban. However, the ABCB1 1236?C?>?T-2677G?>?T-3435C?>?T and 1199?G?>?A SNPs had no significant influence on the intracellular accumulation of rivaroxaban when compared to the wild-type protein. These results suggest that the ABCB1 coding SNPs investigated in the present study are unlikely to contribute to the inter-individual variability in rivaroxaban plasma concentrations.
| S-EPMC6043481 | biostudies-literature
Project description:BackgroundThe methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Loss of supression (Los1) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends replicative lifespan. Herein, a los1-deficient strain of P. pastoris was generated and characterized.MethodsA gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 single-chain variable fragment (scFv) expression were compared between the parent and mutant strains.ResuultsThe los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively.ConclusionThe presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains.
| S-EPMC8334395 | biostudies-literature
Project description:The intracellular penetration of darunavir, a second-generation HIV protease inhibitor, is limited by the activity of the efflux P-glycoprotein (ABCB1). ABCB1 expression and/or activity levels can vary between individuals due to genetic polymorphisms including the c.1199G>A, c.1236C>T, c.2677G>T and c.3435C>T variants, which could in part explain why the pharmacokinetics of darunavir are so variable from one individual to another. While a few clinical studies have failed to demonstrate an influence of these polymorphisms on darunavir pharmacokinetics, drug-drug interactions and methodological limitations may have prevented them from revealing the true influence of ABCB1 variants. In this work, we report on the intracellular accumulation of darunavir in recombinant HEK293 cell lines expressing wild-type ABCB1 or one of several variants: ABCB1 1199A, ABCB1 3435T, and ABCB1 1236T/2677T/3435T. We demonstrate that while ABCB1 expression limits intracellular accumulation of darunavir, there is no significant difference in efflux activity between cells expressing wild-type ABCB1 and those that express any of the studied variants.
| S-EPMC8076219 | biostudies-literature
Project description:Genetically encoded unnatural amino acids provide powerful strategies for modulating the molecular functions of proteins in mammalian cells. However this approach has not been coupled to genome-wide measurements, because efficient unnatural amino acid incorporation is limited to readily transfectable cells and leads to very heterogeneous expression. We demonstrate that rapid piggybac integration of the orthogonal pyrrolysyl-tRNA synthetase (PylS)/tRNAPyl CUA pair (and its derivatives) into the mammalian genome enables efficient, homogeneous unnatural amino acid incorporation into target proteins in diverse cells, and we reveal the distinct transcriptional responses of ES cells and MEFs to amber suppression. Genetically encoding Nε-acetyl-lysine in place of six lysine residues in histone H3, that are known to be post-translationally acetylated, enables deposition of pre-acetylated histones into cellular chromatin, via a synthetic pathway that is orthogonal to enzymatic modification, allowing us to determine the consequences of acetylation at specific amino acids in histones on gene expression. mRNA was sequenced using polyA-enrichment and Truseq library preparation protocol. Two biological replicates were sequences for each cell line and condition
2016-07-03 | E-GEOD-73823 | biostudies-arrayexpress