Molecular Cloning, Sequencing and Expression of a Cdna Coding for Human Antithrombin III

Colau, Brigitte; Cravador, A.; Loriau, R.; Elsen, A Van; Hoylaerts, Marc; Jacobs, P.; Herzog, Albert; Bollen, Alex

doi:10.1016/b978-0-08-033215-4.50040-6

Cited by 3 publications

(4 citation statements)

References 12 publications

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“…Thus, maturation of hATIII becomes a limiting activity above a certain threshold concentration of intracellular hATIII, which is somewhere between 0.5 and 2.1 g hATIII/10 6 cells in this system (Table I). A posttranslational modification of hATIII must occur to some extent to yield active protein, as can be concluded from unsuccessful attempts to express active hATIII in prokaryotic and lower eukaryotic hosts (Bröker et al, 1987;Dingermann et al, 1991;Colau et al, 1985;Prochownik et al, 1983). This indicates that in A11-A279-C7 cells the capability for performing early posttranslational modifications is limited to certain intracellular amounts of a recombinant protein.…”

Section: Discussionmentioning

confidence: 99%

“…Binding of heparin to hATIII induces a conformational change in the hATIII molecule (Einarsson and Andersson, 1977;Fish and Björk, 1979;Olson et al, 1981;Li et al, 1976;Rosenberg and Damus, 1973;Villaneuva and Danishefsky, 1977), which accelerates the reaction between hATIII and thrombin or tightens the heparinantithrombin III complex (Olson et al, 1981;Peterson and Blackburn, 1987). The cDNA for hATIII was cloned (Bock et al, 1982;Chandra et al, 1983;Colau et al, 1985;Prochownik et al, 1983) and stable expression in CHO cells (Wasley et al, 1987;Zettlmeissl et al, 1987) yielded biologically active hATIII (Zettlmeissl et al, 1987). hATIII produced in CHO cells is very similar to plasma hATIII, but shows an altered glycosylation pattern (Zettlmeissl et al, 1989) and affinity for heparin (Björk et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

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Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein

Schröder

Friedl

1997

Biotechnol. Bioeng.

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Overexpression of recombinant proteins in animal cells is commonly achieved by using gene amplification techniques. Gene amplified cells possess up to several thousand genes coding for the target protein. Constitutive expression of these genes leads to high levels of the corresponding mRNA species and the immature protein in the cell. Inefficient processing of these precursors may result from their great abundance in the cell. To study the influence of elevated intracellular levels of a recombinant protein on its maturation and secretion, we examined the maturation and secretion of human antithrombin III (hATIII) in Chinese hamster ovary (CHO) cells at different levels of gene amplification. No loss of vitality was caused by elevated secretion of hATIII. As the intracellular hATIII content increased, the efficiency of hATIII secretion decreased steadily. The state of intracellular hATIII from the different cell lines was studied by determining the specific heparin cofactor activity of hATIII. Intracellular hATIII from the highest amplified cell line displayed a lowered specific heparin cofactor activity indicating the presence of malfolded, only partially folded, or incompletely or incorrectly posttranslationally modified hATIII in this cell line. Thus, the ability of CHO cells to fold and/or introduce posttranslational modifications and subsequently to secrete the recombinant protein becomes saturated, and therefore these processes may become limiting for protein secretion at highly elevated expression levels. This limitation was not due to a general exhaustion of the secretory capacity of the cells because hATIII constituted only a minor fraction of the secreted proteins, even at high expression levels. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 547–559, 1997.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein

Schröder

Friedl

1997

Biotechnol. Bioeng.

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“…Its concentration in plasma is 2.2-2.4 i~mol/l (about 150 Ilg/ml). The antithrombin III gene (Prochownik et al 1985) and cDNA clones have been isolated and characterized (Bock et al 1982;Chandra et al 1983;Colau et al 1985;Prochownik et al 1983). The precursor molecule of antithrombin III comprises a signal peptide of 32 amino acids, which is responsible for secretion of the mature antithrombin III composed of 432 amino acids.…”

Section: Discoideummentioning

confidence: 99%

“…Several groups have succeeded in expressing the human antithrombin III gene in E. coli (Bock et al 1982;Colau et al 1985) and in different cell lines and organisms (Prochownik and Orkin 1984;Stephens et al 1987;Br6ker et al 1987;Zettlmeissl et al 1987). Two groups reported the heterologous expression of biologically active antithrombin III.…”

Section: Discoideummentioning

confidence: 99%

Expression of human antithrombin III in the cellular slime mould Dictyostelium discoideum

Dingermann

Troidl

Bröker

et al. 1991

Appl Microbiol Biotechnol

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In order to test the biotechnological potential of the cellular slime mould Dictyostelium discoideum the cDNA coding for human antithrombin III was expressed in this microorganism. The 1392-bp antithrombin III cDNA was fused to the N-terminal coding part of the D. discoideum actin 6 gene. In constructs carrying this artificial N-terminal coding region only low amounts of antithrombin III were detected. However, constructs from which all actin coding nucleotides were removed produced significant amounts of anti-thrombin III, most of which was secreted into the culture broth. Stationary cultures (1.5 x 10(7) cells/ml) of certain stable transformants accumulated up to 1.0 microgram antithrombin III/ml culture medium within 24 h. The recombinant protein has a slightly smaller molecular weight in sodium dodecyl sulphate-polyacrylamide gels than authentic plasma antithrombin III and it is glycosylated, as determined by concanavalin A labelling.

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Expression of Biologically Active Human Antithrombin III in Chinese Hamster Ovary Cells

1987

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Molecular Cloning, Sequencing and Expression of a Cdna Coding for Human Antithrombin III

Cited by 3 publications

References 12 publications

Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein

Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein

Expression of human antithrombin III in the cellular slime mould Dictyostelium discoideum

Expression of Biologically Active Human Antithrombin III in Chinese Hamster Ovary Cells

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