Enhanced secretion of human granulocyte colony-stimulating factor directed by a novel hybrid fusion peptide from recombinant Saccharomyces cerevisiae at high cell concentration

Cheon Soon Bae, Doo Suk Yang, Ki Ryong Chang, Baik Lin Seong, Jeewon Lee

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Abstract

The synthesis and secretion of recombinant human granulocyte colony- stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombinant Saccharomyces cerevisiae, and some important characteristics of the secreted rhG-CSF are demonstrated. Transcription of the recombinant gene is regulated by a GALl-10 upstream activating sequence (UAS(G)), and the rhG-CSF is expressed in a hybrid fusion protein consisting of signal sequence of Kluyveromyces lactis killer toxin and N-terminal 24 amino acids of human interleukin 1β. The intracellular KEX2 cleavage leads to excretion of mature rhG-CSF into extracellular culture broth, and the cleavage process seems to be highly efficient. In spite of relatively low copy number the plasmid propagation is stably maintained even at nonselective culture conditions. The rhG-CSF synthesis does not depend on galactose level, whereas the production of extracellular rhG-CSF was significantly enhanced by increasing the inducer concentration above a certain level and also by supplementing the nonionic surfactant to the culture medium, which is notably due to the enhanced secretion efficiency. Various immunoblotting analyses demonstrate that none of the rhG-CSF is accumulated in the cell wall fraction and that a significant amount of intracellular rhG-CSF antibody-specific immunoreactive proteins is located in the ER. A core N-glycosylation at fused IL-1β fragment is likely to play a critical role in directing the high-level secretion of rhG-CSF, and the O- glycosylation of secreted rhG-CSF seems nearly negligible. Also the extracellular rhG-CSF is observed to exist as various multimers, and the nature of molecular interaction is evidently not the covalent disulfide bridges. The CD spectra of purified rhG-CSF and Escherichia coli-derived standard show that the conformations of both are similar and are almost identical to that reported for natural hG-CSF.

Original languageEnglish
Pages (from-to)600-609
Number of pages10
JournalBiotechnology and Bioengineering
Volume57
Issue number5
DOIs
Publication statusPublished - 1998 Mar 5

Fingerprint

Glycosylation
Granulocyte Colony-Stimulating Factor
Yeast
Peptides
Saccharomyces cerevisiae
Fusion reactions
Proteins
Molecular interactions
Nonionic surfactants
Transcription
Antibodies
Escherichia coli
Conformations
Amino acids
Genes
Cells
Interleukin-1
Batch Cell Culture Techniques
Protein Sorting Signals
Galactose

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

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title = "Enhanced secretion of human granulocyte colony-stimulating factor directed by a novel hybrid fusion peptide from recombinant Saccharomyces cerevisiae at high cell concentration",
abstract = "The synthesis and secretion of recombinant human granulocyte colony- stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombinant Saccharomyces cerevisiae, and some important characteristics of the secreted rhG-CSF are demonstrated. Transcription of the recombinant gene is regulated by a GALl-10 upstream activating sequence (UAS(G)), and the rhG-CSF is expressed in a hybrid fusion protein consisting of signal sequence of Kluyveromyces lactis killer toxin and N-terminal 24 amino acids of human interleukin 1β. The intracellular KEX2 cleavage leads to excretion of mature rhG-CSF into extracellular culture broth, and the cleavage process seems to be highly efficient. In spite of relatively low copy number the plasmid propagation is stably maintained even at nonselective culture conditions. The rhG-CSF synthesis does not depend on galactose level, whereas the production of extracellular rhG-CSF was significantly enhanced by increasing the inducer concentration above a certain level and also by supplementing the nonionic surfactant to the culture medium, which is notably due to the enhanced secretion efficiency. Various immunoblotting analyses demonstrate that none of the rhG-CSF is accumulated in the cell wall fraction and that a significant amount of intracellular rhG-CSF antibody-specific immunoreactive proteins is located in the ER. A core N-glycosylation at fused IL-1β fragment is likely to play a critical role in directing the high-level secretion of rhG-CSF, and the O- glycosylation of secreted rhG-CSF seems nearly negligible. Also the extracellular rhG-CSF is observed to exist as various multimers, and the nature of molecular interaction is evidently not the covalent disulfide bridges. The CD spectra of purified rhG-CSF and Escherichia coli-derived standard show that the conformations of both are similar and are almost identical to that reported for natural hG-CSF.",
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Enhanced secretion of human granulocyte colony-stimulating factor directed by a novel hybrid fusion peptide from recombinant Saccharomyces cerevisiae at high cell concentration. / Bae, Cheon Soon; Yang, Doo Suk; Chang, Ki Ryong; Seong, Baik Lin; Lee, Jeewon.

In: Biotechnology and Bioengineering, Vol. 57, No. 5, 05.03.1998, p. 600-609.

Research output: Contribution to journalArticle

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