- A Recombinant Human GM-CSF Protein Expressed as an Inclusion form in Escherichia coli Stimulates Colony Formation and Cell Proliferation in vitro
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Ah Young Lee , Jin-Kyoo Kim , Hye Kyung Chung , Eun Kyong Bae , Jung Suk Hwang , Chung Won Cho , Dong Seok Lee , Jae Yong Han , Choon-Taek Lee , Soon-
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J. Microbiol. 2002;40(1):77-81.
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 Abstract
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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematopoietic growth factor involved in the development of myeloid cells from bone marrow, and an activator of mature myeloid cells functioning in a variety of antimicrobial and inflammatory responses. Recently, recombinant GM-CSF is increasingly under clinical study for treatment of various diseases including cancer, infectious diseases and hematopoietic diseases as well as for an immune response modulator. In this study, we constructed a recombinant human GM-CSF (rhGM-CSF) expression plasmid with a pelB leader sequence and His.Tag under T7 promoter control. The expression construct was shown to produce a recombinant protein of 20 kDa in the 8M urea preparation, indicating the rhGM-CSF may be expressed as an insoluble inclusion form. The 20 kDa recombinant protein in 8M urea was transformed into the water-soluble form by dialysis against PBS buffer (phosphate buffered saline). The soluble rhGM-CSF protein was shown to stimulate colony formation and cell proliferation in vitro, indicating that the rhGM-CSF could be refolded into its native form to show colony stimulating activity.
- Continuous Synthesis of Escherichia coli GroEL at a High Temperature
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Young Hak Kwak , Kyong Sun Lee , Ji Yeon Kim , Dong Seok Lee , Han Bok Kim
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J. Microbiol. 2000;38(3):145-149.
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Abstract
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GroEL is a typical molecular chaperone. GroEL synthesis patterns at various culture temperatures in Escherichia coli were investigated in this study. No significant differences in the amount of GroEL produced from the chromosome were found at 30 and 37 C. However, GroEL production increased 3.4-fold at 42 C. GroEL synthesis was not transient but continuous at 42 C, although most heat shock gene expression is known to be transient. To understand the role of the groEL structural gene, a groE promoter-lacZ fusion was constructed. Interestingly, while transcriptional fusion is not thermally inducible, it is inducible by ethanol, suggesting that the secondary structure of the groEL transcript is involved in thermal regulation of the groEL gene. Secondary structures of groE mRNA at 37 and 42 C were compared using the computer program RNAdraw. Distinct structures at the two temperatures were found, and these structures may be related to a high level of GroEL expression at 42 C.
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