Faculty Opinions recommendation of Ebola virus VP35 induces high-level production of recombinant TPL-2-ABIN-2-NF-κB1 p105 complex in co-transfected HEK-293 cells.

2013 ◽  
Author(s):  
Dario Alessi
Keyword(s):  
Ebola Virus ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
High Level ◽  
Level Production

scholarly journals Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

2013 ◽  
Vol 452 (2) ◽  
pp. 359-365 ◽  
Author(s):  
Thorsten Gantke ◽  
Sabrina Boussouf ◽  
Julia Janzen ◽  
Nicholas A. Morrice ◽  
Steven Howell ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Ebola Virus ◽  
Tumour Progression ◽  
Nuclear Factor Κb ◽  
Virus Protein ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dependent Protein ◽  
High Level

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells.

scholarly journals Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels

1998 ◽  
Vol 336 (3) ◽  
pp. 647-658 ◽  
Author(s):  
Theodor CHITLARU ◽  
Chanoch KRONMAN ◽  
Menachem ZEEVI ◽  
Michal KAM ◽  
Adrian HAREL ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Anion Exchange ◽  
Acid Content ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sialic Acid Content ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
High Level

Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver β-galactoside α-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t½β, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.

scholarly journals Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽  
2013 ◽  
Vol 9 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Patience Musiwaro ◽  
Matthew Smith ◽  
Maria Manifava ◽  
Simon A. Walker ◽  
Nicholas T. Ktistakis
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
Author(s):  
Kevin J. Gingrich ◽  
Son Tran ◽  
Igor M. Nikonorov ◽  
Thomas J. Blanck
Keyword(s):  
Charge Transfer ◽  
Calcium Channels ◽  
Dependent Manner ◽  
Current Time ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

2007 ◽  
Vol 9 (4) ◽  
pp. 475-485 ◽  
Author(s):  
R. M. Johann ◽  
Ch. Baiotto ◽  
Ph. Renaud
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Calcium Induces Expression of Cytoplasmic Gelsolin in SH-SY5Y and HEK-293 Cells

2010 ◽  
Vol 35 (7) ◽  
pp. 1075-1082 ◽  
Author(s):  
Lina Ji ◽  
Abha Chauhan ◽  
Ved Chauhan
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Cytoplasmic Gelsolin

Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells

2007 ◽  
Vol 454 (3) ◽  
pp. 441-450 ◽  
Author(s):  
Christian Barmeyer ◽  
Jeff Huaqing Ye ◽  
Shafik Sidani ◽  
John Geibel ◽  
Henry J. Binder ◽  
...  
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Abstract 5316: hERG 1b as a Potential Target for Inherited and Acquired Long QT Syndrome

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Gail A Robertson ◽  
Harinath Sale ◽  
David Tester ◽  
Thomas J O’Hara ◽  
Pallavi Phartiyal ◽  
...  
Keyword(s):  
Action Potential ◽  
Long Qt Syndrome ◽  
Time Course ◽  
Drug Sensitivity ◽  
Long Qt ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Acquired Long Qt Syndrome ◽  
293 Cells ◽  
Qt Syndrome

Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long QT syndrome. Biochemical studies show that native I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homo-oligomers of the original hERG 1a isolate. The hERG 1a and 1b subunits are identical except at the amino (NH2) terminus, which in hERG 1b is much shorter and has a unique primary sequence. We compared the biophysical properties of currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We found that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a two-fold increase in the apparent rates of activation and recovery from inactivation, which reduces rectification and facilitates current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the two channel types. Depending on the action potential model used, loss of 1b predicts an increase in action potential duration of 27 ms (7%) or 41 ms (17%), respectively. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding four-fold positive shift in the IC 50 . Differences in current kinetics and drug sensitivity were modeled by “NH2 mode” gating with conformational states bound by the amino terminus in hERG 1a homomers but not 1a/1b heteromers. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients and absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells nearly eliminated 1b protein. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr , prolong QT interval and enhance drug sensitivity, thus representing a potential mechanism underlying inherited or acquired LQTS.

COOH-terminal association of human smooth muscle calcium channel Cav1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

2007 ◽  
Vol 293 (6) ◽  
pp. C1983-C1990 ◽  
Author(s):  
Minho Kang ◽  
Gracious R. Ross ◽  
Hamid I. Akbarali
Keyword(s):  
Smooth Muscle ◽  
Calcium Channel ◽  
Tyrosine Phosphorylation ◽  
Src Kinase ◽  
Sh2 Domain ◽  
Calcium Currents ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Binding Domains ◽  
293 Cells

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Cav) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCav1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCav1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y2134 prevented SH2 binding and resulted in reduced phosphorylation of hCav1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y1837, Y1861, and Y2134 were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y1837-2134F. These data demonstrate that the COOH terminus of hCav1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

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