Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae.
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 and ion-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 x 106 colony-forming units/mg in the final purified product.
Chen
Jian
J
Institute of Biochemistry, Zhejiang Sci-Tech University, Hangzhou, China.
Nie
Zuo-Ming
ZM
Lü
Zheng-Bing
ZB
Zhu
Cheng-Gang
CG
Xu
Cheng-Zhi
CZ
Jin
Yong-Feng
YF
Wu
Xiang-Fu
XF
Zhang
Yao-Zhou
YZ
D000818:Animals; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism*; D005591:Chemical Fractionation / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011679:Pupa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014462:Ultrafiltration / Q000379:methods
D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor