[Construction and expression of huGM-CSF (9-127)-IL-6(29-184) fusion protein gene].
OBJECTIVE
To construct and express huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity and both huGM-CSF and huIL-6 biologic activities.


METHODS
The novel gene coding for the fusion protein of huGM-CSF(9-127)-IL-6(29-184) was constructed by strategy of step by step cloning in pBV220 expression vector. The amino acids 1-8 of huGM-CSF and the amino acids 1-28 of huIL-6 were deleted by PCR technique. The mutant huGM-CSF (9-127) and huIL-6 (29-184) cDNAs were linked via a linker sequence coding 15 amino acid residues (G-G-S-G-S)3. Fusion protein was expressed in E.coli host strain DH5 alpha. To obtain the fusion protein, Q Sepharose H.P. ion exchange chromatography and Sephacryl S-200 gel filtration were performed. The biologic activities were detected by MTT method.


RESULTS
Fusion protein was expressed in E.coli host strain DH5 alpha in the form of inclusion body. The expression level was more than 25% of the total cell lysate. Through Q Sepharose H.P. ion exchange chromatography and Sephacryl S-200 gel filtration, huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity was obtained. The protein showed both huGM-CSF and huIL-6 biologic activities. The specific activity of huGM-CSF was 1.08 x 10(8) U/mg, and for huIL-6, it reached 1.95 x 10(7) U/mg.


CONCLUSIONS
huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity and both huGM-CSF and huIL-6 biologic activities was obtained.
Sun
Q M
QM
Department of Molecular Biology, Institute of Medical Biology, CAMS, PUMC, Kunming 650118, China.
Xu
W M
WM
Ma
Y B
YB
Yang
X
X
Liu
H Y
HY
Sun
M S
MS
Dai
C B
CB
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D015850:Interleukin-6 / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor