[Expression, purification and identification of mouse granulocyte-macrophage colony-stimulating factor].
OBJECTIVE
To construct mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) expression vector and express, purify and refold mGM-CSF protein.


METHODS
Based on previously constructed fusion protein hIL-2/mGM-CSF expression vector, pET-11c/mGM-CSF expression vector was constructed routinely and transformed into BL21 (DE3). The inclusion body protein was washed with our patented method and refolded with renaturation buffer containing low-concentration guanidinium chloride (Gu.Cl). The refolded protein was purified with affinity chromatography.


RESULTS
pET-11c/mGM-CSF vector was constructed successfully. The host bacteria was cultured in TH broth and induced with 0.1 mmol/L IPTG at 32 degrees C, which resulted in the expression level of 60.6%. The best refolding effect was achieved with the renaturation media containing glutathione and 1.5 mol/L Gu.HCl. After purification with affinity chromatography, the purity of the target mGM-CSF protein reached 95% with activity of 5x10(6) U/mg.


CONCLUSIONS
Engineered bacteria BL21/pET- 11c/mGM-CSF with efficient mGM-CSF expression and laboratory scale renaturation and purification of mGM-CSF have been established, which facilitates further researches into the anti-tumor function of the dendritic cells and GM-CSF in vivo.
Wen
Qian
Q
Institute of Molecular Immunology, School of Biotechnology, Southern Medical University, Guangzhou 510515, China. wencaoxi@163.com
Ma
Li
L
Su
Jin
J
Luo
Wei
W
Wang
Xiao-ning
XN
D000818:Animals; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor