Authors: Chen, Jian¹; Nie, Zuo-Ming¹; Lü, Zheng-Bing¹; Zhu, Cheng-Gang²; Xu, Cheng-Zhi¹; Jin, Yong-Feng²; Wu, Xiang-Fu³; Zhang, Yao-Zhou⁴

Source: Applied Biochemistry and Biotechnology, Volume 141, Number 1, April 2007 , pp. 149-159(11)

Publisher: Humana Press

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.

Keywords: Bombyx mori nucleopolyhedrovirus; silkworm pupae; human granulocyte-macrophage colony-stimulating fa; purification; isoelectric precipitation; (NH4)2SO4 fractional precipitation; target protein

Document Type: Research article

DOI: http://dx.doi.org/10.1007/s12010-007-9217-1

Affiliations: 1: 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, 4: Institute of Biochemistry, Zhejiang Sci-Tech University, 310018, Hangzhou, China, Email: yaozhou@zist.edu.cn

Publication date: 2007-04-01

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• In this Subject: Anatomy & Physiology ,  Biotechnology ,  Organic Chemistry ,  Biochemistry
• By this author: Chen, Jian ;  Nie, Zuo-Ming ;  Lü, Zheng-Bing ;  Zhu, Cheng-Gang ;  Xu, Cheng-Zhi ;  Jin, Yong-Feng ;  Wu, Xiang-Fu ;  Zhang, Yao-Zhou

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