Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor.
To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest. In this study, human granulocyte-macrophage colony-stimulating factor was successfully expressed in silkworm pupae using B. mori nucleopolyhedrovirus, purified and characterized with respect to its physico-chemical properties. The target protein expressed had an apparent molecular mass of 29 kDa and an isoelectric point of 5.1. The protein was purified using three chromatographic steps with a final recovery of 10.3%. Finally, approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4 x 10(6) cfu mg(-1). The results of this study suggest that silkworm pupae represent a convenient and low-cost bioreactor for the expression of heterologous proteins.
Chen
Jian
J
College of Life Sciences, Zhejiang University, Hangzhou 310029, China.
Wu
Xiang-Fu
XF
Zhang
Yao-Zhou
YZ
D000818:Animals; D019149:Bioreactors; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011679:Pupa / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor