RESUMEN
In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD+) under the hypothesis that NAD+ regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD+ treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD+ precursors - nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) - were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss-Handler pathways for NAD+ biosynthesis was verified, and then the effect of NAD+ precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD+ levels by up to 70.6% compared to the non-treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD+ treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD+ treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD+ precursor-treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD+ precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD+ precursors as medium and feed components for the biopharmaceutical production.
Asunto(s)
Cricetulus , NAD , Niacinamida , Células CHO , Animales , NAD/metabolismo , Niacinamida/metabolismo , Niacinamida/análogos & derivados , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Mononucleótido de Nicotinamida/metabolismo , Niacina/metabolismo , Compuestos de Piridinio/metabolismo , Cricetinae , Técnicas de Cultivo de Célula/métodos , Anticuerpos Monoclonales/metabolismo , Glucosa/metabolismoRESUMEN
Identification of recombinant gene integrations sites in the Chinese hamster ovary (CHO) cell genome is increasingly important to assure monoclonality. While next-generation sequencing (NGS) is commonly used for the gene integration site analysis, it is a time-consuming and costly technique as it analyzes the entire genome. Hence, simple, easy, and inexpensive methods to analyze transgene insertion sites are necessary. To selectively capture the integration site of transgene in the CHO genome, we applied splinkerette-PCR (spPCR). SpPCR is an adaptor ligation-based method using splinkerette adaptors that have a stable hairpin loop. Restriction enzymes with high frequencies in the CHO genome were chosen using a Python script and used for the in vitro spPCR assay development. After testing on two CHO housekeeping genes with known loci, the spPCR-based genome walking technique was successfully applied to recombinant CHO cells to identify the transgene integration site. Finally, the comparison with NGS methods exhibited that the time and cost required for the analysis can be substantially reduced. Taken together, the established technique would aid the stable cell line development process by providing a rapid and cost-effective method for transgene integration site analysis.
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Genoma , Cricetinae , Animales , Cricetulus , Células CHO , Transgenes , Genoma/genética , Reacción en Cadena de la PolimerasaRESUMEN
Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. KEY POINTS: ⢠Polyamine supplementation enhanced cell growth and production in a dose-dependent manner ⢠Polyamine type and concentration in the media affected mAb quality ⢠Optimizing polyamines in the media is suggested in CHO cell bioprocessing.
Asunto(s)
Poliaminas , Espermidina , Cricetinae , Animales , Poliaminas/farmacología , Poliaminas/metabolismo , Células CHO , Cricetulus , Espermidina/metabolismo , Putrescina/farmacología , Putrescina/metabolismo , Espermina/metabolismo , Espermina/farmacología , Proliferación CelularRESUMEN
Aerobic glycolysis and its by-product lactate accumulation are usually associated with adverse culture phenotypes such as poor cell viability and productivity. Due to the lack of knowledge on underlying mechanisms and accompanying biological processes, the regulation of aerobic glycolysis has been an ongoing challenge in culture process development for therapeutic protein productivity. Nicotinamide adenine dinucleotide (NAD+ ), a coenzyme and co-substrate in energy metabolism, promotes the conversion of inefficient glycolysis into an efficient oxidative phosphorylation (OXPHOS) pathway. However, the effect of NAD+ on Chinese hamster ovary (CHO) cells for biopharmaceutical production has not been reported yet. In this work, we aimed to elucidate the influence of NAD+ on cell culture performance by examining metabolic shifts and mAb productivity. The supplementation of NAD+ increased the intracellular concentration of NAD+ and promoted SIRT3 expression. Antibody titer and the specific productivity in the growth phase were improved by up to 1.82- and 1.88-fold, respectively, with marginal restrictions on cell growth. NAD+ significantly reduced the accumulation of reactive oxygen species (ROS) and the lactate yield from glucose, determined by lactate accumulation versus glucose consumption (YLAC/GLC ). In contrast, OXPHOS capacity and amino acid consumption rate increased substantially. Collectively, these results suggest that NAD+ contributes to improving therapeutic protein productivity in bioprocessing via inducing an energy metabolic shift.
Asunto(s)
Glucosa , NAD , Cricetinae , Animales , Cricetulus , NAD/metabolismo , Células CHO , Glucosa/metabolismo , Ácido Láctico/metabolismo , Suplementos DietéticosRESUMEN
Methotrexate (MTX)-mediated gene amplification has been widely used in Chinese hamster ovary (CHO) cells for the biomanufacturing of therapeutic proteins. Although many studies have reported chromosomal instability and extensive chromosomal rearrangements in MTX-mediated gene-amplified cells, which may be associated with cell line instability issues, the mechanisms of chromosomal rearrangement formation remain poorly understood. We tested the impact of DNA double-strand breaks (DSBs) on chromosomal rearrangements using bleomycin, a DSB-inducing reagent. Bleomycin-treated CHO-DUK cells, which are one of the host cell lines deficient in dihydrofolate reductase (Dhfr) activity, exhibited a substantial number of cells containing radial formations or non-radial formations with chromosomal rearrangements, suggesting that DSBs may be associated with chromosomal rearrangements. To confirm the causes of DSBs during gene amplification, we tested the effects of MTX treatment and the removal of nucleotide base precursors on DSB formation in Dhfr-deficient (i.e., CHO-DUK) and Dhfr-expressing (i.e., CHO-K1) cells. Immunocytochemistry demonstrated that MTX treatment did not induce DSBs per se, but a nucleotide shortage caused by the MTX-mediated inhibition of Dhfr activity resulted in DSBs. Our data suggest that a nucleotide shortage caused by MTX-mediated Dhfr inhibition in production cell lines is the primary cause of a marked increase in DSBs, resulting in extensive chromosomal rearrangements after gene amplification processes.
RESUMEN
Chinese hamster ovary (CHO) cell line instability and clonality issues can affect cell culture phenotypes such as cell growth, productivity, or product quality and remain challenges for biopharmaceutical manufacturing. While there have been efforts for characterizing cell line instability in CHO production cell lines, a pre-existing level of cell line instability in CHO host cells has not been determined. In this study, cell line instability and chromosomal heterogeneity of the host, CHO-DUK cell line, is reported by using a karyotyping approach. Long-term cultures and karyotype analysis of CHO-DUK cells revealed that the growth rate was higher in later passage cultures, correlating with an increase in the population ratio containing the mar3 chromosome. To further investigate a correlation between growth rate and karyotype, CHO-DUK cells are subcloned by limiting dilution and the growth rate and karyotype of each subclone are determined. Subclones containing the mar3 chromosome exhibit higher cell growth rates than subclones without the mar3 chromosome. Finally, karyotype analysis indicate that CHO-DUK cells, as well as limiting-diluted subclones, exhibit a karyotypically heterogeneous population, suggesting that chromosomal rearrangements occur spontaneously and frequently even in non-engineered host cells. These results demonstrate CHO host cell line instability and suggest that chromosomal instability and karyotypic changes are associated with compromised clonality (heterogeneity), affecting cell line (in)stability in CHO host cells.
Asunto(s)
Células CHO , Inestabilidad Cromosómica/genética , Evolución Clonal/genética , Heterogeneidad Genética , Animales , Línea Celular , Linaje de la Célula/genética , Cricetinae , Cricetulus , CariotipificaciónRESUMEN
Chinese hamster ovary (CHO) cells, the major mammalian host cells for biomanufacturing of therapeutic proteins, have been extensively investigated to enhance productivity and product quality. However, cell line instability resulting in unexpected changes in productivity or product quality continues to be a challenge. Based on previous reports about causes and characteristics of production instability, we hypothesized that chromosomal rearrangements due to genomic instability are associated with production instability and that these events can be characterized. We developed a production instability model using secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) as well as a framework to quantify chromosomal rearrangements by karyotyping. In the absence of methotrexate (MTX), CHO-SEAP cells exhibited a slightly increased growth rate, a significantly decreased specific productivity, and changes in the chromosomal rearrangement ratio of seven chromosomes. In contrast, when MTX was re-introduced, the growth rate and SEAP productivity reversed to the initial values, demonstrating the reversibility of production instability in CHO-SEAP cells. Fluorescence in situ hybridization analysis identified that the SEAP genes were incorporated in the chromosomal rearrangement (insertion) part of the der(Z9) chromosome. Karyotype analysis indicated that the insertion ratio of the der(Z9) chromosome decreased in the CHO-SEAP cells grown without MTX, demonstrating a correlation between chromosomal rearrangement and production instability. Our results support a mechanism for production instability, wherein a randomly generated chromosomal rearrangement (or genotype) results in cells with a growth advantage that is also associated with non (or low)-producing traits. As a result, the non-producing cells grow faster and thereby outgrow the producing population. Biotechnol. Bioeng. 2017;114: 1045-1053. © 2016 Wiley Periodicals, Inc.
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Reactores Biológicos/normas , Células CHO , Cariotipo , Cariotipificación/métodos , Análisis de la Célula Individual/métodos , Animales , Línea Celular , Cricetinae , CricetulusRESUMEN
Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity â¼100-fold and the heparin/heparan sulfate yield â¼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in â¼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of â¼90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.
Asunto(s)
Anticoagulantes , Células CHO , Heparina/biosíntesis , Ingeniería Metabólica , Animales , Reactores Biológicos , Vías Biosintéticas , Cricetinae , Cricetulus , Heparina/metabolismoRESUMEN
Chinese hamster ovary (CHO) cells are a major host cell line for the production of therapeutic proteins, and CHO cell and Chinese hamster (CH) genomes have recently been sequenced using next-generation sequencing methods. CHOgenome.org was launched in 2011 (version 1.0) to serve as a database repository and to provide bioinformatics tools for the CHO community. CHOgenome.org (version 1.0) maintained GenBank CHO-K1 genome data, identified CHO-omics literature, and provided a CHO-specific BLAST service. Recent major updates to CHOgenome.org (version 2.0) include new sequence and annotation databases for both CHO and CH genomes, a more user-friendly website, and new research tools, including a proteome browser and a genome viewer. CHO cell-line specific sequences and annotations facilitate cell line development opportunities, several of which are discussed. Moving forward, CHOgenome.org will host the increasing amount of CHO-omics data and continue to make useful bioinformatics tools available to the CHO community.
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Células CHO , Ingeniería Celular , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Tratamiento Basado en Trasplante de Células y Tejidos , Cricetinae , Cricetulus , Bases de Datos Genéticas , Bases de Datos de Ácidos Nucleicos , Genoma , HumanosRESUMEN
Chinese hamster ovary (CHO) cells are important hosts for the production of therapeutic proteins. Recent genome sequencing studies provide an initial baseline of information useful for understanding cell line performance in terms of product quality attributes. However, the lack of a well-established reference genome together with concerns about genome stability have not yet permitted the community to define the detailed relationship between the genome and cell line performance. Emerging efforts to define a new reference genome, together with new data on genome stability, herald an era where cell line's with defined genomes can be combined with defined process parameters to yield product quality attribute control.
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Células CHO , Cricetulus/genética , Genoma , Análisis de Secuencia de ADN , Animales , Mapeo Cromosómico , Mapeo Contig , Inestabilidad GenómicaRESUMEN
Improvement of Chinese hamster ovary (CHO) cells for therapeutic protein production has great potential for the manufacturing of biopharmaceuticals. This commentary by Baik and Lee discusses the study by Klanert et al., which describes an improved tool that will allow greater control over the design of CHO cells.
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Regulación de la Expresión Génica/genética , Ingeniería Genética/métodos , MicroARNs/genética , MicroARNs/metabolismo , AnimalesRESUMEN
Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities.
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Biotecnología/métodos , Heparina/biosíntesis , Sulfotransferasas/metabolismo , Animales , Anticoagulantes/química , Conformación de Carbohidratos , Línea Celular Tumoral , Heparina/química , Mastocitoma/metabolismo , Ratones , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfotransferasas/genéticaRESUMEN
HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.
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Aparato de Golgi/enzimología , Heparina/biosíntesis , Heparitina Sulfato/biosíntesis , Ingeniería Metabólica , Sulfotransferasas/biosíntesis , Amidohidrolasas/biosíntesis , Amidohidrolasas/genética , Animales , Células CHO , Cricetinae , Cricetulus , Aparato de Golgi/genética , Heparina/genética , Heparitina Sulfato/genética , Humanos , Ratones , Sulfotransferasas/genéticaRESUMEN
Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Since Chinese hamster ovary (CHO) cells are capable of producing heparan sulfate (HS), a related polysaccharide naturally, and heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. We developed stable human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) expressing cell lines based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells. Both activity assay and disaccharide analysis showed that engineered HS attained heparin-like characteristics but not identical to pharmaceutical heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.
RESUMEN
Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.
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Amidohidrolasas/biosíntesis , Expresión Génica , Heparina/biosíntesis , Ingeniería Metabólica , Sulfotransferasas/biosíntesis , Amidohidrolasas/genética , Animales , Células CHO , Cricetinae , Cricetulus , Heparina/genética , Humanos , Ratones , Sulfotransferasas/genética , Transfección/métodos , TransgenesRESUMEN
A high-resolution method for the separation and analysis of disaccharides prepared from heparin and heparan sulfate (HS) using heparin lyases is described. Ultra-performance liquid chromatography in a reverse-phase ion-pairing mode efficiently separates eight heparin/HS disaccharides. The disaccharides can then be detected and quantified using electrospray ionization mass spectrometry. This method is particularly useful in the analysis of small amounts of biological samples, including cells, tissues, and biological fluids, because it provides high sensitivity without being subject to interference from proteins, peptides, and other sample impurities.
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Cromatografía Liquida/métodos , Disacáridos/análisis , Heparina/análogos & derivados , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Células CHO , Camelus , Cricetinae , Cricetulus , Disacáridos/aislamiento & purificación , Heparina/análisis , Heparina/aislamiento & purificación , Liasa de Heparina/metabolismo , Heparina de Bajo-Peso-Molecular/análisis , Heparitina Sulfato/análisisRESUMEN
Bcl-x(L), a member of the Bcl-2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl-x(L) overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl-x(L) expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and MS analysis. The consequences of Bcl-x(L) overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl-x(L) overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif-containing GTPase-activating protein 1), cell proliferation (dihydrolipoamide-S-acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl-x(L) not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects.
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Butiratos/farmacología , Cricetulus/metabolismo , Electroforesis en Gel Bidimensional/métodos , Proteoma/metabolismo , Regulación hacia Arriba , Proteína bcl-X/genética , Animales , Apoptosis/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus/genética , Eritropoyetina/biosíntesis , Regulación de la Expresión Génica , Espectrometría de Masas/métodos , Proteoma/genéticaRESUMEN
Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull-down assay was performed with dual-tagged (N-terminal GST- and C-terminal hexahistidine-tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual-tagged EPO were then resolved by two-dimensional gel electrophoresis (2DE) and identified by MALDI-TOF MS/MS. A total of 27 protein spots including glucose-regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull-down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells.
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Eritropoyetina/química , Proteínas de Choque Térmico/química , Animales , Células CHO , Cricetinae , Cricetulus , Electroforesis en Gel Bidimensional , Eritropoyetina/biosíntesis , Eritropoyetina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Proteómica , Proteínas Recombinantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Ingeniería de TejidosRESUMEN
Sodium butyrate (NaBu) is known to enhance the specific productivity of Chinese hamster ovary cells expressing human thrombopoietin. In order to better understand the intracellular responses of these cells resulting from NaBu treatment, the proteomic profiles of cells treated with various concentrations of NaBu (0-3mM) were compared using two-dimensional electrophoresis (2-DE). Based on spot intensities, 80 high intensity protein spots were selected. Fifty-six of the 80 protein spots, which represent 28 different kinds of proteins, were identified by MALDI-TOF-MS and MS/MS. Compared to control without NaBu treatment, the expression levels of 2 proteins (glucose regulated protein 78 (GRP 78) and peroxiredoxin 4) were increased over two fold with NaBu treatment and the expression level of phosphopyruvate hydratase was decreased over two fold with NaBu treatment. Due to multiplicity (multiple spots for one protein), a change in one single spot intensity from a 2-DE gel image may not represent the total change in expression level for that protein. Western blot analyses of GRP78, HSC70 and ERp57 confirmed the results of the MS analyses. However, a degree of change in expression level differed between the two methods, suggesting the necessity of a validating method to determine the total amount of the protein.