A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein

Asselbergs, Fred A.M.; Will, Hans; Wingfield, Paul T.; Hirschi, Marlis

doi:10.1016/0022-2836(86)90312-8

Cited by 30 publications

(9 citation statements)

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“…In rodent cells, the major transcripts are the 2.0 kb species, the promoter of which is located within the pre-S gene, in agreement with previous reports (Dubois et al, 1980;Christman et al, 1982;Asselsberg et al, 1986). In primate cells, in addition to the 2.0 kb RNA, there is abundant expression of the 2.4 kb RNA, the promoter of which precedes the pre-S region.…”

Section: Discussionsupporting

confidence: 90%

Preferential Expression Of The Large Hepatitis B Virus Surface Antigen Gene by an Adenovirus-hepatitis B Virus Recombinant

Yuasa

Kajino

Saito

et al. 1991

Journal of General Virology

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Section: Discussionsupporting

confidence: 90%

Preferential Expression Of The Large Hepatitis B Virus Surface Antigen Gene by an Adenovirus-hepatitis B Virus Recombinant

Yuasa

Kajino

Saito

et al. 1991

Journal of General Virology

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“…In this plasmid, the expression of foreign DNA is driven by the immediate-early promoter of mouse cytomegalovirus, containing the dihydrofolate reductase (dhfr) gene. The plasmid was used to transfect dhfr-CHO cells (recombinant cell line CHO-CGI-1) [25]. High-producing clones were selected after methotrexate-induced DNA amplification, using ELISA techniques to quantify K,tu-PA [22].…”

Section: Expression Of Ktu-pa Cultivation Of Cho Cells and Isolatimentioning

confidence: 99%

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K₂tu‐PA expressed in Chinese hamster ovary cells

Bergwerff

Oostrum

Asselbergs

et al. 1993

European Journal of Biochemistry

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A recombinant human plasminogen activator hybrid variant KJu-PA, expressed in Chinese hamster ovary cells, is partially glycosylated at Asnl2 (A chain, kringle-2 domain) and completely glycosylated at Asn247 (B chain, protease domain). After release of the N-linked carbohydrate chains by peptide-N4-(N-acetyl-/3-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the protein by gel permeation chromatography, then fractionated by FPLC on Mono Q, followed by HPLC on Lichrosorb-NH,, and analysed by 500-MHz 'H-NMR spectroscopy. The following types of carbohydrates occur : monosialylated diantennary (8 %), disialylated diantennary (45 %), disialylated tri-and tri'-antennary (1 %), trisialylated tri-and tri'-antennary (28 %), and tetrasialylated tetra-antennary (18 %) structures, all having fucose in a(l-g)-linkage at the Asn-bound Nacetylglucosamine. Sialic acid occurred exclusively in a(2-3)-linkage to galactose, and consisted of N-acetylneuraminic acid (94 %), N-glycolylneuraminic acid (3 %), and N-acetyl-9-O-acetylneuraminic acid (3%). In addition, glycopeptide fragments corresponding with the A or B chain of K,tu-PA were analysed. The oligosaccharides attached to Asnl2 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K,tu-PA with that of tissue-type plasminogen activator from different biological sources showed significant differences.Profiling studies on different K,tu-PA production batches demonstrated that the structures of Nlinked oligosaccharides were identical, but that relative amounts vary with the applied isolation procedure of the chimeric glycoprotein.Plasminogen activators (PA) are serine proteases, that convert the inactive proenzyme plasminogen into the active enzyme plasmin, thereby functioning as important initiators of fibrinolysis. Two types of plasminogen activators can be distinguished, namely, the urinary-type (urokinase, u-PA) and the tissue-type (t-PA) [l]. Urokinase and recombinant t-PA are of clinical interest for the treatment of acute vascular diseases like myocardial infarction [2-41. To obtain artery reperfusion, high blood levels of PA are required, because of its rapid clearance by the liver. Such pharmaceutical use is associated with a variable degree of systemic fibrinolytic acCorrespondence to J. P. Kamerling, Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P. 0. Box 80.075, NL-3508 TB Utrecht, The Netherlands Abbreviations. PNGase-F, peptide-P-(N-acetyl-/%glucosaminyl)-asparagine amidase F; PA, plasminogen activator; u-PA, urinarytype plasminogen activator; t-PA, tissue-type plasminogen activator; CHO, Chinese hamster ovary ; NeuSAc, N-acetylneuraminic acid; NeuSGc, N-glycolylneuraminic acid ; Neu5,9Ac2, N-acetyl-9-O-acetylneuraminic acid; lD, one-dimensional ; 2D, two-dimensional; HOHAHA, homonuclear Hartmann-Hahn ; MLEV, composite pulse devised by Malcom Levitt; COSY, scalar shift correlated spectroscopy ; ROESY, rotating-frame nuclear Overhauser enhancement spectroscopy.Enzymes. Peptid...

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“…Fax: /41- (0) ) with the E. coli hygromycin B-phosphotransferase gene (hph expressing the T antigens of SV40, pSF-SVT, was created by in-or hpt) were created by inserting the SV40 origin/SfiI site from pSFserting a second SV40 origin/SfiI site into the BamHI site of pSV81 dhfr into BamHI-cut pSVneo2911 (17) and pCGA25cNAT (6), respec- (17). With the inserted T antigen gene, expression vectors can repli-tively.…”

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“…Thus, the success rate B; n.u., not unique), first a cDNA fragment coding for the (soluble) of the dual-selection method with the SfiI cassettes was extracellular part of the low-affinity IgE receptor (sCD23) (22) was at least a hundred fold higher and only 6 weeks instead inserted (C). The K2tu-PA expression vector was then created by replacing the NcoI and BglII fragment of the sCD23 cDNA with of 6-9 months (3,17) was needed to obtain the higha NcoI-BclI fragment coding for K2tu-PA (D). Cleavage with SfiI producer cell lines.…”

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Gene Cassettes for Directional Insertion at theSfiI Cleavage Site in the SV40 Replication Origin of Mammalian Expression Vectors

Asselbergs

Gandor

Widmer

1996

Analytical Biochemistry

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A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein

Cited by 30 publications

References 38 publications

Preferential Expression Of The Large Hepatitis B Virus Surface Antigen Gene by an Adenovirus-hepatitis B Virus Recombinant

Preferential Expression Of The Large Hepatitis B Virus Surface Antigen Gene by an Adenovirus-hepatitis B Virus Recombinant

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K₂tu‐PA expressed in Chinese hamster ovary cells

Gene Cassettes for Directional Insertion at theSfiI Cleavage Site in the SV40 Replication Origin of Mammalian Expression Vectors

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A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein

Cited by 30 publications

References 38 publications

Preferential Expression Of The Large Hepatitis B Virus Surface Antigen Gene by an Adenovirus-hepatitis B Virus Recombinant

Preferential Expression Of The Large Hepatitis B Virus Surface Antigen Gene by an Adenovirus-hepatitis B Virus Recombinant

Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K2tu‐PA expressed in Chinese hamster ovary cells

Gene Cassettes for Directional Insertion at theSfiI Cleavage Site in the SV40 Replication Origin of Mammalian Expression Vectors

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Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K₂tu‐PA expressed in Chinese hamster ovary cells