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1.
Heterogeneity of Chinese hamster ovary cell-produced recombinant murine interferon-gamma 总被引:3,自引:0,他引:3
Chinese hamster ovary cells transformed with a hybrid expression plasmid containing both the murine interferon-gamma (MuIFN-gamma) and the murine dihydrofolate reductase-coding sequences were subjected to selection in stepwise increasing concentrations of methotrexate. By this procedure the production rate of MuIFN-gamma was increased from an initial level of approximately 20,000 to approximately 500,000 antiviral units per milliliter of culture supernatant. [35S]Methionine-labeled proteins secreted by these cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without prior immunoprecipitation with polyclonal or monoclonal antibodies against splenocyte-derived MuIFN-gamma. Besides two major components of Mr 19,000 and 38,000, a multiplicity of minor components were immunoprecipitated. Cells treated with tunicamycin, an inhibitor of N-glycosylation, secrete two major components of Mr 14,000 and 27,000 and only two minor components of Mr 12,000 and 13,000. When the proteins were labeled with [35S]cysteine, a residue that is only present at the carboxyl terminus of the mature MuIFN-gamma, no minor components could be detected in the growth medium of tunicamycin-treated cells. The presented results indicate that the heterogeneity of the recombinant Chinese hamster ovary-produced MuIFN-gamma is due to at least three cumulative modifications of the Mr 14,000 MuIFN-gamma peptide: carboxyl-terminal proteolytic processing (the Mr 13,000 and 12,000 components), variations in N-glycosylation (components ranging in size from Mr 12,000 to 26,500), and dimerization (components ranging from Mr 27,000 to 50,000). 相似文献
2.
3.
Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells 总被引:11,自引:0,他引:11
Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimentation equilibrium experiments showed the recombinant erythropoietin preparation to be essentially a single macromolecular component with a molecular weight of 30,400 and a carbohydrate content of 39%. The Stokes radius of recombinant erythropoietin was estimated to be 32 A from gel filtration, much larger than the 20-A radius calculated for a sphere of the observed molecular weight. This difference may be ascribed to the extensive glycosylation. The fluorescence and phosphorescence spectra showed that the luminescent tryptophan(s) is (are) solvent-exposed and can be quenched by I- and acrylamide but not by Cs+. On acid titration, the recombinant erythropoietin showed a conformational transition with a midpoint of pH 4.1. This suggests that the net charges on the protein moiety rather than on the whole molecule play a role in protein structure stability. 相似文献
4.
The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc. 相似文献
5.
Structure of recombinant human interleukin 5 produced by Chinese hamster ovary cells 总被引:4,自引:0,他引:4
Y Minamitake S Kodama T Katayama H Adachi S Tanaka M Tsujimoto 《Journal of biochemistry》1990,107(2):292-297
The complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure. Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis. After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence. The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86. In addition, we concluded that Thr-3 and Asn-28 were glycosylated. The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate. 相似文献
6.
Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines. 总被引:5,自引:0,他引:5
N Moguilevsky L Garcia-Quintana A Jacquet C Tournay L Fabry L Piérard A Bollen 《European journal of biochemistry》1991,197(3):605-614
The cDNA encoding human myeloperoxidase carries three ATG codons in frame; 144, 111 and 66 bp upstream from the proprotein DNA sequence. In order to determine the most efficient signal sequence, three cDNA modules starting at each of the ATG were cloned into an eucaryotic expression vector and stably expressed in Chinese hamster ovary cell lines. In all three cases, recombinant human myeloperoxidase (recMPO) was secreted into the culture medium of transfected cells, indicating that each of the signal peptides functions efficiently. One of the recombinant cell lines, which was amplified using methotrexate, overexpresses enzymatically active recMPO up to 6 micrograms.ml-1.day-1. The recombinant product was purified by a combination of ion-exchange and metal-chelate chromatography, and characterized in terms of molecular mass, amino-terminal amino acid analysis, glycosylation, physicochemical properties and biological activity. The data show that recMPO is secreted essentially as a monomeric, heme-containing, single-chain precursor of 84 kDa which exhibits peroxidase activity. Amino-terminal analysis indicated that cleavage of the signal peptide occurs between amino acids 48 and 49. In addition, recMPO appeared to be glycosylated up to the last stage of sialylation, to an extent similar to that of the natural enzyme. Specific activity measurements as well as stability data, in various pH, temperature, ionic strength and reducing conditions, indicated that the recombinant single-chain enzyme behaves essentially in the same way as the natural two-chain molecule. Finally, recMPO was shown to exert potent cytotoxicity towards Escherichia coli when provided with its physiological substrates, i.e. hydrogen peroxide and chloride ions. 相似文献
7.
M N Lioubin L Madisen H Marquardt R Roth K S Kovacina A F Purchio 《Journal of cellular biochemistry》1991,45(1):112-121
Latent recombinant transforming growth factor-beta 2 (LrTGF-beta 2) complex has been purified from serum-free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF-beta 2 (414) precursor. Under neutral conditions, LrTGF-beta 2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro-region sequences. Acidification of LrTGF-beta 2 resulted in the release of mature 24 kDa TGF-beta 2 from the high molecular weight pro-region-containing complex, suggesting that TGF-beta 2 was non-covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF-beta 2. Protein sequence analysis of the purified TGF-beta 2 pro-region indicated that signal peptide cleavage occurred between ser(20) and leu(21). The pro-region, which previously was found to contain mannose-6-phosphate, bound to the mannose-6-phosphate receptor. Proteolytic cleavage of mature TGF-beta 2 from pro-TGF-beta 2 was inhibited by monensin and chloroquine suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF-beta 2 precursor. 相似文献
8.
Expression and purification of recombinant human Angiopoietin-1 produced in Chinese hamster ovary cells 总被引:1,自引:0,他引:1
Hwang SJ Kim HZ Koh GY Lee GM 《In vitro cellular & developmental biology. Animal》2007,43(5-6):162-167
Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation. In an effort to produce large quantities of Ang1,
recombinant Chinese hamster ovary (rCHO) cells expressing a high level of recombinant human Ang1 protein (rhAng1) with an
amino terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO
cells and subsequent gene amplification in a medium containing step-wise increments of methotrexate, such as 0.02, 0.08, and
0.32 μM. The rhAng1 secreted from rCHO cells was purified at a purification yield of 18.4% from the cultured medium using
an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng1 as heterogeneous
multimers. Moreover, rhAng1 expressed in rCHO cells is biologically active in vitro as demonstrated by its ability to bind
to the Tie2 receptor and to phosphorylate Tie2. Therefore, the rhAng1 produced from CHO cells could be useful for clarifying
the biological effects of exogenous rhAng1 in the future. 相似文献
9.
Hwang SJ Choi HH Kim KT Hong HJ Koh GY Lee GM 《Protein expression and purification》2005,39(2):14410-183
Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future. 相似文献
10.
Korekane H Korekane A Yamaguchi Y Kato M Miyamoto Y Matsumoto A Hasegawa T Suzuki K Taniguchi N Ookawara T 《Glycoconjugate journal》2011,28(3-4):183-196
Extracellular superoxide dismutase (EC-SOD), the major SOD isoenzyme in biological fluids, is known to be N-glycosylated and heterogeneous as was detected in most glycoproteins. However, only one N-glycan structure has been reported in recombinant human EC-SOD produced in Chinese hamster ovary (CHO) cells. Thus, a precise N-glycan profile of the recombinant EC-SOD is not available. In this study, we report profiling of the N-glycan in the recombinant mouse EC-SOD produced in CHO cells using high-resolution techniques, including the liberation of N-glycans by treatment with PNGase F, fluorescence labeling by pyridylamination, characterization by anion-exchange, normal and reversed phase-HPLC separation, and mass spectrometry. We succeeded in identifying 26 different types of N-glycans in the recombinant enzyme. The EC-SOD N-glycans were basically core-fucosylated (98.3% of the total N-glycan content), and were high mannose sugar chain, and mono-, bi-, tri-, and tetra-antennary complex sugar chains exhibiting varying degrees of sialylation. Four of the identified N-glycans were uniquely modified with a sulfate group, a Lewis(x) structure, or an α-Gal epitope. The findings will shed new light on the structure-function relationships of EC-SOD N-glycans. 相似文献
11.
Inoue N Watanabe T Kutsukake T Saitoh H Tsumura H Arai H Takeuchi M 《Glycoconjugate journal》1999,16(11):707-718
Human thrombopoietin (TPO) that regulates the numbers of megakaryocytes and platelets is a heavily N- and O-glycosylated glycoprotein hormone with partial homology to human erythropoietin (EPO). We prepared recombinant human TPO produced in Chinese hamster ovary (CHO) cells and analyzed the sugar chain structures quantitatively using 2-aminobenzamide labeling, sequential glycosidase digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS).We found bi-, tri- and tetraantennary complex-type sugar chains with one or two N-acetyllactosamine repeats, which are common to recombinant human EPO produced in CHO cells. On the other hand, there were triantennary sugar chains with one or two N-acetyllactosamine repeats that were specific to the recombinant human TPO, and their distributions of branch structures were also different. These results suggested that proximal protein structure should determine the branch structure of Asn-linked sugar chains in addition to the glycosyltransferases subset. 相似文献
12.
为了对工程中国仓鼠卵巢(CHO)细胞所产人源重组促红素(rhEPO)的N-糖基化特点进行考察,静置培养工程细胞后,通过等电聚焦和凝集素共沉淀对培养上清中的rhEPO进行分析,并对无血清培养上清中乳酸脱氢酶(LDH)和唾液酸酶活性进行检测,发现这株CHO细胞可以表达唾液酸含量较高的rhEPO蛋白。但是随着培养时间的延长,细胞的存活率逐渐降低,死亡的细胞将胞内的唾液酸酶释放到胞外,唾液酸酶的降解作用会造成N-糖链分枝末端的唾液酸占有率降低,导致rhEPO蛋白糖基化形态的变化。所使用的方法及得到的结果为进一步对工业过程进行分析提供了参考。 相似文献
13.
Recombinant human granulocyte-colony-stimulating factor (G-CSF) was purified from Chinese hamster ovary cells transfected with human G-CSF cDNA. The recombinant human G-CSF was treated with alkaline borohydride and the oligosaccharide-alditols liberated were fractioned by gel filtration on a Bio-Gel P-4 column, followed by high-performance liquid chromatography by use of a strong anion exchanger. Two oligosaccharide-alditols were obtained and their structures were identified by component analysis and 500-MHz 1H-NMR spectroscopy. The structures of the sugar chains were NeuAc alpha 2-3Gal beta 1-3GalNAcol and NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAcol. 相似文献
14.
Young Kue Han Yeon‐Gu Kim Jee Yon Kim Gyun Min Lee 《Biotechnology and bioengineering》2010,105(6):1187-1192
During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310–610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase‐3, caspase‐7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3‐II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310–610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality. Biotechnol. Bioeng. 2010;105: 1187–1192. © 2009 Wiley Periodicals, Inc. 相似文献
15.
Chinese hamster ovary cell lines are good manufacturing practice-certified host cells and are widely used in the field of biotechnology to produce therapeutic antibodies. Recombinant protein productivity in cells is strongly associated with cell growth. To control cell proliferation, many approaches have previously been tested including: genetic engineering, chemical additives such as cell cycle inhibitors, and temperature shift of the culture. To be widely adopted in the biopharmaceutical industry, the culture methods should be simple, uniform and safe. To this end, we examined the use a natural compound to improve the production capacity. In this study, we focused on the antioxidants, catechin polyphenols, which are found in green tea, for cell proliferation control strategies. (–)-Epigallocatechin-3-gallate (EGCG), the major catechin that induces G0/G1 cell cycle arrest, was investigated for its effect on recombinant protein production. Adding EGCG to the cell culture media resulted in slower cellular growth and longer cell longevity, which improved the specific productivity and total yield of recombinant IgG1 in batch cultures by almost 50% for an extra 2 or 3 days of culture. A lower l-glutamine consumption rate was observed in cells cultured in EGCG-containing media, which may be suggesting that there was less stress in the culture environment. Additionally, EGCG did not affect the N-glycan quality of IgG1. Our results indicated that adding EGCG only on the first day of the culture enhanced the specific productivity and total amount of recombinant protein production in batch cultures. This approach may prove to be useful for biopharmaceutical production. 相似文献
16.
Joon Chul Lee Do Yun Kim Duk Jae Oh Ho Nam Chang 《Biotechnology and Bioprocess Engineering》2008,13(5):560-565
A rCHO cell line of DUKX origin 26*-320, producing recombinant antibody against the human platelet, was cultivated in a two-stage
depth filter perfusion system (DFPS) for 20 days in order to attain high recombinant antibody concentration. The productivity
of the first stage DFPS bioreactor reached 53 times that of the batch culture in a controlled stirred tank reactor and was
showed 12.1 mg/L antibody concentration at a perfusion rate of 6.0 d−1. Glucose concentration in the first DFPS was maintained at 1.5 g/L to avoid cell damage in the perfusion culture. A second
stage DFPS system was attached to the first DFPS, which resulted in a low glucose concentration of 0.02 g/L and a high antibody
concentration of 23.9 mg/L. The two-stage depth filter perfusion culture yielded 60% higher product concentration than the
batch and 49-fold higher productivity of 69.3 mg/L/d in comparison with that (1.4 mg/L/d) in a batch system. Furthermore,
antibody concentration of the second stage was 97% higher than that of the first stage, and the antibody productivities were
comparable to that of the first stage. This two-stage DFPS system also showed potential for higher titer production of recombinant
antibody and high volumetric productivity for long-term culture of bio-pharmaceutical substances. 相似文献
17.
Sato Y Liu C Wojczyk BS Kobata A Spitalnik SL Endo T 《Biochimica et biophysica acta》1999,1472(1-2):344-358
The N- and O-glycans of recombinant amyloid precursor protein (APP), purified from Chinese hamster ovary cells transfected with the human 695-amino acid form of APP, were separately released by hydrazinolysis under different conditions. The reducing ends of the released N- and O-glycans were reduced with NaB3H4 and derivatized with 2-aminobenzamide (2AB), respectively. After acidic N-glycans were obtained by anion-exchange column chromatography, these were converted to neutral oligosaccharides by sialidase digestion, demonstrating that their acidic nature was entirely due to sialylation. The sialidase-treated N-glycans were then fractionated by lectin column chromatography and their structures were determined by linkage-specific sequential exoglycosidase digestion. These results demonstrated that recombinant APP has bi- and triantennary complex type N-glycans with fucosylated and nonfucosylated trimannosyl cores. In a similar fashion, the 2AB-labeled O-glycans derived from APP were determined to be mono- and disialylated core type 1 structures. Taken together, these results indicate that recombinant APP has sialylated bi- and triantennary N-glycans with fucosylated and nonfucosylated cores and sialylated O-glycans with core type 1 structures. 相似文献
18.
Since sialic acid content is known to be a critical determinant of the biological properties of glycoproteins, it is essential to characterize and monitor sialylation patterns of recombinant glycoproteins intended for therapeutic use. This study reports site- and branch-specific differences in sialylation of human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture. Sialylation profiles were quantitated by reversed-phase HPLC separations of the site-specific pools of tryptic glycopeptides representing IFN-gamma's two potential N-linked glycosylation sites (i.e., Asn(25) and Asn(97)). Although sialylation at each glycosylation site was found to be incomplete, glycans of Asn(25) were more heavily sialylated than those of Asn(97). Furthermore, Man(alpha1-3) arms of the predominant complex biantennary structures were more favorably sialylated than Man(alpha1-6) branches at each glycosylation site. When the sialylation profile was analyzed throughout a suspension batch culture, sialic acid content at each site and branch was found to be relatively constant until a steady decrease in sialylation was observed coincident with loss of cell viability. The introduction of a competitive inhibitor of sialidase into the culture supernatant prevented the loss of sialic acid after the onset of cell death but did not affect sialylation prior to cell death. This finding indicated that incomplete sialylation prior to loss of cell viability could be attributed to incomplete intracellular sialylation while the reduction in sialylation following loss of cell viability was due to extracellular sialidase activity resulting from cell lysis. Thus, both intracellular and extracellular processes defined the sialic acid content of the final product. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 390-398, 1977. 相似文献
19.
Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression
Condon RG Schaefer EJ Santoro M Longley R Tsao YS Zurawski SM Liu Z 《Biotechnology progress》2003,19(1):137-143
Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins. 相似文献
20.
A Chinese Hamster Ovary cell line, CHO1-15500, producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification
system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral
SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the
lag (0.065 pg cell−1 h−1) and early decline (0.040 pg cell−1 h−1) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic
cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase.
The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity
of 0.080 pg c-h−1. Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant
tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms
are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant
cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between
expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization
of the animal cell production system. This information facilitates rational process design, including operating mode, modelling
and control, and medium formulation. 相似文献