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1.
High-yield production of recombinant antibody fragments in HEK-293 cells using sodium butyrate 总被引:1,自引:0,他引:1
To develop new recombinant monoclonal antibody fragments for therapy and imaging, it is indispensable to have a simple and easy procedure to handle the eukaryotic expression system for production of proteins in high amounts. Gene amplification techniques such as the dehydrofolate reductase (DHFR) system in Chinese hamster ovary cells or the glutamine synthase system in myeloma cells have a couple of disadvantages. The selection procedure is complex, time-consuming, and not fruitful in all cases. The toxic drug methotrexate (for the DHFR system) can increase the production rate but decreases the specific growth rate of the cells. The production rate is not always stable over a long-term cultivation period. To overcome these problems, we are using stably transfected human embryonic kidney (HEK-293) cells in combination with an efficient screening method. Sodium butyrate can increase the expression of recombinant antibody fragments in the transfectomas up to 500 micrograms/4.2 x 10(7) cells/24 h corresponding to 175 micrograms/mL culture medium. This strategy allows a rapid development of new recombinant monoclonal antibody fragments and allows one to proceed rapidly to in vivo testing. 相似文献
2.
Lithium chloride (LiCl), which induces cell cycle arrest at G2/M phase, is known as a specific production rate (q p)-enhancing additive in recombinant Chinese hamster ovary (CHO) cell culture. To determine the potential of LiCl as a chemical additive that enhances transient gene expression (TGE), LiCl was added to the CHO-NK and human embryonic kidney 293E (HEK293E) cell cultures before and/or after transfection with polyethylenimine as a transfection reagent. The effect of this addition on transfection efficiency (pre-treatment) and q p enhancement during TGE (post-treatment) was examined. For the TGE of monoclonal antibody (mAb) in CHO-NK cells, pretreatment alone with 10 mM LiCl and post-treatment alone with 5 mM LiCl resulted in 1.2- and 3.4-fold increase of maximum mAb concentration (MMC), respectively, compared with the TGE without LiCl treatment. Furthermore, combinatorial treatment with LiCl (10 mM for pre-treatment and 5 mM for post-treatment) synergistically increased the TGE of mAb (5.3-fold increase in MMC). Likewise, combinatorial treatment with LiCl (10 mM for pre-treatment and 15 mM for post-treatment) in HEK293E cells synergistically increased the TGE of mAb (4.9-fold increase in MMC). Taken together, the data obtained here demonstrate that combinatorial treatment with LiCl is a useful means to improve TGE in CHO as well as HEK293 cells. 相似文献
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For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in
the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein
production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth
arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures
with 2 mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture.
For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild
hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia
accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in
the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells
when applying conditions that reduce or arrest the growth of these cells. 相似文献
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There is an imperative need for expression systems allowing the efficient and robust manufacturing of high quality glycoproteins. In the present work, HEK-293 cells stably expressing interferon-α2b were further engineered with the insertion of the yeast pyruvate carboxylase 2 gene. In batch cultures, marked reductions in lactate and ammonia production were observed compared to the parental cell clone. Although the maximum specific growth rate remained unchanged, the altered metabolism led to a 2-fold increase in maximum cell density and 33% increase in the integral of viable cell concentration and interferon production yield. The underlying metabolic changes were further investigated using various 13C-labeled substrates and measuring the resulting lactate mass isotopomer distributions. Simultaneous metabolite and isotopomer balancing allowed the accurate determination of key intracellular fluxes. Such detailed and quantitative knowledge about the central carbon metabolism of the cells is instrumental to further support the development of high-yield fed-batch processes. 相似文献
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Summary Arsenic is an established human carcinogen. The role of aquaglyroporins (AQPs) in arsenic disposition was recently identified. In order to examine whether organic anion transporting polypeptide-C (OATP-C) also plays a role in arsenic transport, OATP-C cDNA was transfected into cells of a human embryonic kidney cell line (HEK-293). Transfection increased uptake of the model OATP-C substrate, estradiol-17β-D-glucuronide, by 10-fold. In addition, we measured uptake and cytotoxicity of arsenate, arsenite, monomethylarsonate(MMAV), and dimethylarsinate (DMAV). Transfection of OATP-C increased uptake and cytotoxicity of arsenate and arsenite, but not of MMAV or DMAV. Rifampin and taurocholic acid (a substrate of OATP-C) reversed the increased toxicity of arsenate and arsenite seen in OATP-C-transfected cells. The increase in uptake of inorganic arsenic was not as great as that of estradiol-17β-D-glucuronide. Our results suggest that OATP-C can transport inorganic arsenic in a (GSH)-dependent manner. However, this may not be the major pathway for arsenic transport. 相似文献
8.
Expression of Drosophila trehalose-phosphate synthase in HEK-293 cells increases hypoxia tolerance 总被引:5,自引:0,他引:5
Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has not been feasible thus far. The disaccharide trehalose, which accumulates dramatically during heat shock, enhances thermotolerance and reduces aggregation of denatured proteins. Previous studies from our laboratory showed that over-expression of Drosophila trehalose-phosphate synthase (dtps1) increases the trehalose level and anoxia tolerance in flies. To determine whether trehalose can protect against anoxic injury in mammalian cells, we transfected the dtps1 gene into human HEK-293 cells using the recombinant plasmid pcDNA3.1(-)-dtps1 and obtained more than 20 stable cell strains. Glucose starvation in culture showed that HEK-293 cells transfected with pcDNA3.1(-)-dtps1 (HEK-dtps1) do not metabolize intracellular trehalose, and, interestingly, these cells accumulated intracellular trehalose during hypoxic exposure. In contrast to HEK-293 cells transfected with pcDNA3.1(-) (HEK-v), cells with trehalose were more resistant to low oxygen stress (1% O2). To elucidate how trehalose protects cells from anoxic injury, we assayed protein solubility and the amount of ubiquitinated proteins. There was three times more insoluble protein in HEK-v than in HEK-dtps1 after 3 days of exposure to low O2. The amount of Na+-K+ ATPase present in the insoluble proteins dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas there was no significant change in HEK-dtps1 cells. Ubiquitinated proteins increased dramatically in HEK-v cells after 2 and 3 days of exposure but not in HEK-dtps1 cells over the same period. Our results indicate that increased trehalose in mammalian cells following transfection by the Drosophila tps1 gene protects cells from hypoxic injury. The mechanism of this protection is likely related to a decrease in protein denaturation, through protein-trehalose interactions, resulting in enhanced cellular recovery from hypoxic stress. 相似文献
9.
Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines. To overcome these bottlenecks, we have explored the use of various growth factors to enhance specific productivity and titers in the context of transient gene expression. For that purpose, several growth factors were cloned and screened for their effect on transient gene expression in HEK293E and CHO-DG44 cells. In particular, acidic fibroblast growth factor (aFGF) was able to increase specific productivity by 60% and recombinant protein titers by 80% in HEK293E cells, while FGF9 increased titers by 250% in CHO-DG44 cells. 相似文献
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Aulestia FJ Redondo PC Rodríguez-García A Rosado JA Salido GM Alonso MT García-Sancho J 《The Biochemical journal》2011,435(1):227-235
Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms. 相似文献
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Recombinant human Factor IX (rFIX) was cloned in a mammalian expression vector and transfected into CHO and HEK-293. Treatment
with 10−9 M methyl testosterone increased rFIX production by 30–50% in CHO and HEK clones. However, 10−9 M 17β-oestradiol increased production of rFIX by ~50% in CHO-F7 clone and decreased production by 48% and 37% in CHO-F8 and
HEK-F2-6, respectively. Progesterone treatment inhibited rFIX production in both cell lines. Production of rFIX can thus be
increased by sex hormone treatment and therefore used to enhance biotechnological production in mammalian cells. 相似文献
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Phosphate transport by the human renal cotransporter NaPi-3 expressed in HEK-293 cells 总被引:1,自引:0,他引:1
The human renal Na-PO4cotransporter gene NaPi-3 was expressed in human embryonic kidneyHEK-293 cells, and the transport characteristics were measured in cellstransfected with a vector containing NaPi-3 or with the vector alone(sham transfected). The initial rate of32PO4influx had saturation kinetics for external Na andPO4 with K Na1/2 of 128 mM(PO4 = 0.1 mM) andK PO41/2of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfectedcells expressing the transporter. Transfection had no effect on theNa-independent 32PO4influx, but transfection increased Na-dependent32PO4influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenateinhibited flux with an inhibition constant of 0.4 mM. The phosphatetransport in sham- and NaPi-3-transfected cells has nearly the sametemperature dependence in the absence and presence of extracellularNa. The Na-dependent phosphate flux decreased with pH insham-transfected cells but was pH independent in transfected cells. TheNa-dependent32PO4influx was inhibited byp-chloromercuriphenylsulfonate,phosphonoformate, phloretin, vanadate, and5-(N-methyl-N-isobutyl)-amiloridebut not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior ofNaPi-3 homologues in the renal tubule of other species and, thus,demonstrate the fidelity of this transfection system for the study ofthis protein. Commensurate with the increased functional expression,there was an increase in the amount of NaPi-3 protein by Westernanalysis. 相似文献
14.
Kamp TJ Hu H Marban E 《American journal of physiology. Heart and circulatory physiology》2000,278(1):H126-H136
The activity of native L-type Ca channels can be facilitated by strong depolarizations. The cardiac Ca channel alpha(1C)-subunit was transiently expressed in human embryonic kidney (HEK-293) cells, but these channels did not exhibit voltage-dependent facilitation. Coexpression of the Ca channel beta(1a)- or beta(2a)-subunit with the alpha(1C)-subunit enabled voltage-dependent facilitation in 40% of cells tested. The onset of facilitation in alpha(1C) + beta(1a)-expressing HEK-293 cells was rapid after a depolarization to +100 mV (tau = 7.0 ms). The kinetic features of the facilitated currents were comparable to those observed for voltage-dependent relief of G protein inhibition demonstrated for many neuronal Ca channels; however, intracellular dialysis with guanosine 5'-O-(2-thiodiphosphate) and guanosine 5'-O-(3-thiotriphosphate) in the patch pipette had no effect on facilitation. Stimulation of G protein-coupled receptors, either endogenous (somatostatin receptors) or coexpressed (adenosine A(1) receptors), did not affect voltage-dependent facilitation. These results indicate that the cardiac Ca channel alpha(1C)-subunit can exhibit voltage-dependent facilitation in HEK-293 cells only when coexpressed with an auxiliary beta-subunit and that this facilitation is independent of G protein pathways. 相似文献
15.
The production of milligram quantities of purified, active, folded membrane protein from heterologous expression systems remains a general challenge due to intrinsically low expression levels, misfolding, and instability. Here we report the overexpression and purification of milligram quantities of functional Saccharomyces cerevisiae G-protein-coupled receptor, Ste2p, from transiently transfected human embryonic kidney 293 EBNA1 cells. Fluorescent microscopy indicates localization of Ste2p-GFP and Fc-Ste2p-GFP fusion receptors to the cell membrane. Up to 2 mg (approximately 10 pmol/million cells) of the Fc-Ste2p-GFP fusion and 1 mg of a Ste2p-Strep-TagII/(His)8-tagged version were purified per liter of culture following protein A-Sepharose and Talon metal affinity chromatography, respectively. Two distinct fluorescent labels, the hydrophobic 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) and the more hydrophilic fluorescein-5-maleimide (FM), were individually attached to the C-terminus of the alpha-mating factor ligand by addition of a reactive cysteine residue to produce active fluorescent pheromones. In vitro fluorescent ligand binding assays demonstrated that a high percentage of the recombinant purified receptor is correctly folded and able to bind ligand. KD values of 34 +/- 3 and 300 +/- 20 nM were observed respectively for the CPM- and FM-labeled ligands. These results combined with blue-shifted emission peaks and loss of fluorescent quenching observed for both fluorescent-labeled Cys alpha-factors when bound to receptor support a model in which the C-terminus of the ligand is packed in a hydrophobic pocket at the interface between the transmembrane and extracellular loop domains. Overall, we present an efficient system for recombinant production of milligram quantities of purified Ste2p in a biologically active form with applications to future structure and functional studies. 相似文献
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Transient gene expression in suspension HEK-293 cells: application to large-scale protein production
Baldi L Muller N Picasso S Jacquet R Girard P Thanh HP Derow E Wurm FM 《Biotechnology progress》2005,21(1):148-153
Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r-protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r-proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. The third method, entirely serum-free, has been successfully applied to both suspension-adapted CHO and HEK-293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK-293 EBNA cells of some out of more than 20 secreted r-proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5-10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12-well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch-to-batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins. 相似文献
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Pilar Alcalá Neus Ferrer-Miralles Jordi X. Feliu Antonio Villaverde 《Biotechnology letters》2002,24(19):1543-1551
The biophysical nature of the signal transduction in enzymatic biosensors through which the paratope-epitope interaction enhances enzyme activity, is essentially unknown. Fab fragments of efficiently activating antibodies are, in general, poor sensor activators suggesting that the bivalent antibody binding could contribute to the sensing process. We have cloned and produced in E. coli a recombinant SD6 scFv fragment directed against a sensing peptide, displayed on a model -galactosidase-based biosensor. While the enzymatic response to scFv-binding is not detectable, the simultaneous presence of scFv and the poorly-activator SD6 Fab fragment results in a non-additive, efficient sensor response, enhancing the enzyme activity up to about 200%. This co-operative effect, which is also observed by combining SD6 scFv and the non-homologous anti-peptide 4C4 Fab, confirms that the enzyme up-regulation requires multiple and probably heterogeneous contacts between the sensor molecule and the analytes, but not necessarily done by bivalent molecular ligands. 相似文献
19.
Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins. 相似文献
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Brini M Manni S Pierobon N Du GG Sharma P MacLennan DH Carafoli E 《The Journal of biological chemistry》2005,280(15):15380-15389
Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders. 相似文献