Effects of gene dosage on the expression of human growth hormone cDNA in Escherichia coli

Yamakawa, Minoru; Sugisaki, Kiyoko; Morimoto, Makoto; Tanaka, Minoru; Yamamoto, Masanori; Ichikawa, Tadashi; Nakashima, Kenichiro

doi:10.1016/0167-4781(89)90095-x

Cited by 7 publications

(3 citation statements)

References 16 publications

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“…Guarna et al (1995) have shown that an optimal cDNA gene dosage exists for the transient production of activated protein C (APC) in CHO cells. This data is very similar to data reported on the influence of plasmid content in Escherichia coli (Aiba et al, 1982;Bailey et al, 1983;Bailey, 1985, 1986;Yamakawa et al, 1989) on protein production and shows that the productivity achievable by gene amplified mammalian cells is limited by steps involved in gene expression.…”

Section: Introductionsupporting

confidence: 90%

Untitled

1999

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The elevation of expression levels for secreted glycoproteins by gene amplification in mammalian cells shows a saturation behavior at high levels of gene amplification. At high expression levels a drop in the secretion efficiency for the recombinant protein occurs (Schröder and Friedl, 1997), coinciding with the appearance of misfolded protein in the cell. In this communication we investigated whether additional limitations exist at the levels of transcription and translation. Four Chinese hamster ovary (CHO) cell lines expressing different amounts of human antithrombin III (ATIII) were used as a model system. A tenfold increase in the ATIII cDNA copy number from the lowest to the highest producing cell line coincided with a 38-fold increase in ATIII mRNA levels, and an 80-fold increase in the amount of intracellular ATIII levels. The data was analyzed using a simple kinetic model. The following conclusions were derived: I. The transcriptional activity for the recombinant protein is not saturated. II. Translation itself is not saturated either, but may be downregulated as secretion efficiency drops. III. Two explanations for the previously reported drop in secretion efficiency for the recombinant protein with increasing expression level are possible: A. Protein degradation is an alternative fate for translated ATIII and the fraction of ATIII degraded after translation increases as expression level is increased. B. Translation is downregulated as the secretory apparatus becomes exhausted to maintain cell viability.

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Section: Introductionsupporting

confidence: 90%

Untitled

1999

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“…For E. coli it is well established that the overall gene expression efficiency is maximal at an optimal plasmid copy number per cell and then decreases as the number of plasmid copies per cell increases (Aiba et al, 1982;Bailey et al, 1983;Bailey, 1985, 1986;Yamakawa et al, 1989). The plasmid content is dependent on the growth rate of E. coli, and high plasmid copy numbers inhibit the formation of proteins and the growth of E. coli (Bailey et al, 1983;Bailey, 1985, 1986;Yamakawa et al, 1989). In contrast, only limited information is available for the effect of cloned gene dosage on the production of heterologous proteins in mammalian cells.…”

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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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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“…The luciferase gene from firefly, Photinus pyralis (de Wet et al, 1987), and the hGH gene (Yamakawa et al, 1989) were inserted into pBM050, a transfer plasmid vector (Maeda, 1989). The transfer vectors (pBmPL, pBmhGH) were purified by caesium chloride density-gradient centrifugation.…”

Section: Construction Of Recombinant Virusesmentioning

confidence: 99%

Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.

Suzuki¹,

Kanaya

Okazaki

et al. 1997

Journal of General Virology

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Infection by a baculovirus (Bombyx mori nuclear polyhedrosis virus, BmNPV) in silkworm (Bombyx mori) larvae is highly efficient as an expression system for the production of useful proteins. However, the amount of the protein of interest expressed tends to decrease in the later stages of infection presumably due, in part, to a proteinase produced in the larval haemolymph. The N-terminal amino acid sequence of a proteinase purified from the haemolymph of BmNPV-infected larvae was identical to the internal amino acid sequence of the viral cysteine proteinase gene of BmNPV, suggesting that the cysteine proteinase in the haemolymph originated from the BmNPV gene. We constructed a

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Effects of gene dosage on the expression of human growth hormone cDNA in Escherichia coli

Cited by 7 publications

References 16 publications

Untitled

Untitled

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

Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.

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