Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in <Emphasis Type="Italic">Bombyx mori</Emphasis> pupae |
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Authors: | Jian Chen Zuo-Ming Nie Zheng-Bing Lü Cheng-Gang Zhu Cheng-Zhi Xu Yong-Feng Jin Xiang-Fu Wu Yao-Zhou Zhang |
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Institution: | (1) Present address: Institute of Biochemistry, Zhejiang Sci-Tech University, 310018 Hangzhou, China;(2) College of Life Sciences, Zhejiang University, 310029 Hangzhou, China;(3) Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 200031 Shanghai, China |
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Abstract: | Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) acts on many different kinds of cells, including monocytes,
macrophages, granulocytes, eosinophils, and multipotential stem cells. To explore further explore pharmaceutical action, we
expressed hGM-CSF by the Bombyx mori nucleopolyhedrovirus expression system in silkworm pupae. However, purifying recombinant proteins from silkworm pupae on
a large scale has been a big challenge. To establish purification methods suitable for mass production, we tried two crude
preparation methods: (NH4)2SO4 fractional precipitation and isoelectric precipitation with a combination of gel filtration andion-exchange chromatography.
The isoelectric precipitation method was found to be more efficient. With this method, we eventually obtained approx 11.7
mg of 95% pure product from 1000 g of infected silkworm pupae. The recovery of purified protein was greatly increased, by
approx 40%, compared with the other method. The biologic activity of this protein was determined up to 9.0×106 colony-forming units/mg in the final purified product. |
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Keywords: | Bombyx mori nucleopolyhedrovirus silkworm pupae human granulocyte-macrophage colony-stimulating factor purification isoelectric precipitation (NH4)2SO4 fractional precipitation target protein |
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