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Cloning and Expression of Bioactive Human Granulocyte Colony Stimulating Factor in Pichia Pastoris
Author(s) -
Chien SuFang
Publication year - 2010
Publication title -
journal of the chinese chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.329
H-Index - 45
eISSN - 2192-6549
pISSN - 0009-4536
DOI - 10.1002/jccs.201000118
Subject(s) - pichia pastoris , granulocyte colony stimulating factor , chemistry , recombinant dna , pichia , intracellular , receptor , cloning (programming) , microbiology and biotechnology , biochemistry , pharmacology , gene , biology , genetics , chemotherapy , computer science , programming language
For over ten years, recombinant human Granulocyte Colony Stimulating Factor (hG‐CSF) has been broadly used in clinical applications. Many sources of G‐CSF have the disadvantage of being in intracellular or aggregated form. In this paper, we report the molecular cloning and gene expression in the protease resistant strain of yeast (SMD1168). More than 90% of the G‐CSF protein is in secreted form and about 100 mg of the biologic active human G‐CSF can be obtained from two liters of bioreactor. The recombinant G‐CSF promotes HL‐60 (Human promyelocytic cell) cell proliferation and elevates the mitochondrial succinate dehydrogenase activity. The dose effects of rG‐CSF on cell proliferation responses to the total numbers of the receptors on the cell surfaces have been investigated and the quantitative results were obtained. The receptors are saturated by G‐CSF at the 50 ng/mL for 1 × 10 5 cells. The prompt increasing in the succinate dehydrogenas activity was also observed. This effect is comparable to that from the commercial source. We have successfully cloned and expressed a bioactive rhG‐CSF from the medium in the Pichia pastoris system. This specific product is expected to meet the clinical applications in the treatments of neutropenia, acute cerebral ischemia and other neuro‐degenerated diseases.

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