Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells

Hacker, David L.; Kiseljak, Divor; Rajendra, Yashas; Thurnheer, Sarah; Baldi, Lucia; Wurm, Florian M.

doi:10.1016/j.pep.2013.09.001

Cited by 66 publications

(53 citation statements)

References 139 publications

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“…Transient transfection was performed following a protocol modified from Hacker et al (19) using PEI (1 mg/ml, 25-kDa linear PEI, Polysciences). One day before the transfection, the cells were seeded at a density of 5 ϫ 10 5 /ml.…”

Section: Transfection and Expression Of Epo-s126v And Other Expressinmentioning

confidence: 99%

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Yang

Wang

2016

Journal of Biological Chemistry

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Understanding the biosynthetic pathway of protein glycosylation in various expression cell lines is important for controlling and modulating the glycosylation profiles of recombinant glycoproteins. We found that expression of erythropoietin (EPO) in a HEK293S N-acetylglucosaminyltransferase I (GnT I) ؊/؊ cell line resulted in production of the Man 5 GlcNAc 2 glycoforms, in which more than 50% were core-fucosylated, implicating a clear GnT I-independent core fucosylation pathway. Expression of GM-CSF and the ectodomain of Fc␥IIIA receptor led to ϳ30% and 3% core fucosylation, suggesting that the level of core fucosylation also depends on the nature of the recombinant proteins.To elucidate the GnT I-independent core fucosylation pathway, we generated a stable HEK293S GnT I ؊/؊ cell line with either knockdown or overexpression of FUT8 by a highly efficient lentivirus-mediated gene transfer approach. We found that the EPO produced from the FUT8 knockdown cell line was the pure Man 5 GlcNAc 2 glycoform, whereas that produced from the FUT8-overexpressing cell line was found to be fully core-fucosylated oligomannose glycan (Man 5 GlcNAc 2 Fuc). These results provide direct evidence that FUT8, the mammalian ␣1,6-fucosyltransferase, is the sole enzyme responsible for the GnT I-independent core fucosylation pathway. The production of the homogeneous core-fucosylated Man 5 GlcNAc 2 glycoform of EPO in the FUT8-overexpressed HEK293S GnT I ؊/؊ cell line represents the first example of production of fully core-fucosylated high-mannose glycoforms.

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Section: Transfection and Expression Of Epo-s126v And Other Expressinmentioning

confidence: 99%

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Yang

Wang

2016

Journal of Biological Chemistry

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“…WT opsin, and G90V and N55K mutants were constructed on a synthetic bovine opsin gene in the pMT4 vector 33 . The WT, and the G90V and N55K opsin mutants were transiently expressed in COS-1 cells by chemical transfection using PEI reagent as previously described 34,35 . After 48 h, the cells were harvested and regenerated with 10 μM 9-cis-retinal in solvent buffer with overnight incubation.…”

Section: Expression Purification and Preparation Of Rho And Recombimentioning

confidence: 99%

Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa

et al. 2015

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Mutations in the visual photoreceptor rhodopsin are the cause of the retinal degenerative disease retinitis pigmentosa. Some naturally occurring mutations can lead to protein conformational instability. Two such mutations, N55K and G90V, in the first and second transmembrane helices of the receptor, have been associated with sector and classical retinitis pigmentosa, respectively, and showed enhanced thermal sensitivity. We have carefully analyzed the effect of phospholipid bicelles on the stability and ligand binding properties of these two mutants and compared it with those of the detergent-solubilized samples. We have used a phospholipid bilayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2dihexanoyl-sn-glycero-3-phosphocholine (DHPC). We find that DMPC/DHPC bicelles dramatically increase the thermal stability of the rhodopsin mutants G90V and N55K. The chromophore stability and regeneration of the mutants were also increased in bicelles when compared to their behavior in a dodecyl maltoside detergent solution. The retinal release process was slowed in bicelles, and chromophore entry, after illumination, was improved for the G90V mutant but not for N55K. Furthermore, fluorescence spectroscopy measurements showed that bicelles allowed more exogenous retinal binding to the photoactivated G90V mutant than in a detergent solution. In contrast, N55K could not reposition any chromophore either in the detergent or in bicelles. The results demonstrate that DMPC/DHPC bicelles can counteract the destabilizing effect of the disease-causing mutations and can modulate the structural changes in the ensuing receptor photoactivation in a distinct specific manner for different retinitis pigmentosa mutant phenotypes.

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“…However, stable cell line development is a time-consuming process taking several months to complete (Browne and Al-Rubeai, 2007;Matasci et al, 2008;Wurm, 2004). For rapid access to recombinant proteins, transient gene expression (TGE) with suspension-adapted mammalian cells is often employed (Geisse and Voedisch, 2012;Hacker et al, 2013;Pham et al, 2006). Recent improvements in TGE in CHO, through host cell engineering and bioprocess optimization, have resulted in recombinant protein yields up to 2 g/L (Cain et al, 2013;Daramola et al, 2014;Rajendra et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools

Balasubramanian¹,

Matasci²,

Kadlecova

et al. 2015

Journal of Biotechnology

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Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells

Cited by 66 publications

References 139 publications

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa

Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools

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