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Solubilization and
purification of Escherichia coli expressed GST-fusion
human vascular endothelial growth factors with
N-Lauroylsarcosine
Jian-ping Zou1,2,
Jing Xu3, Licheng Liu4, Shuxiang Li3,
Chunfu Wu1, Guanhua Du1*
1Department
of Pharmacology, Shenyang Pharmaceutical University,
Shenyang, P.R. China
110016.
2Beijing
JPJX Bio-Med Co. Ltd, Beijing, P.R. China 100085.
3Department
of Immunology, National Vaccine and Serum Institute, 4
Sanjianfang Nanli, Beijing, P. R. China 100024.
4Department
of Biochemistry, College of Life Sciences, Lanzhou
University,
Lanzhou, P. R. China 730000.
*Corresponding author. E-mail:
dugh@imm.ac.cn. Tel.:
++861063165184. Fax: ++861063165184.
Accepted
3 March, 2009 |
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Vascular endothelial growth factor (VEGF) is a potent mitogen
for tumor angiogenesis. Clinically, VEGF detection in human
blood can be expected to be used in the very near future for
cancer screening, prognosis, monitoring of therapy and
diagnosis. VEGF has been identified as the target for the
treatment of cancer. Though prokaryotic expression of VEGF
has been done, the solubilization and purification is time
consuming and empirical. In this study, VEGF165
and VEGF121 were cloned into pGEX-4T-1 vector,
and GST-VEGF fusion proteins were expressed in
Escherichia coli at 37°C. The inclusion bodies of
GST-VEGF fusion proteins were solubilized with N-Lauroylsarcosine
(sarkosyl).
Briefly, the cell suspension with inclusion body was added
with sarkosyl at a final concentration of 1.5%. After the
disruption of cells, the clarified supernatant
containing sarkosyl was added with Triton X-100 at a final
concentration of 3%. The GST-VEGFs were
purified by
affinity
chromatography on glutathione Sepharose 4B. The overall
yield was approximately 10 – 12 mg/l cell culture. The
binding assay showed that the GST-VEGF165 binds
to VEGF receptor in a dose dependent manner.
The current work provides a novel procedure for
solubilization and purification of GST-VEGF fusion proteins,
and no laborious procedures for separation of inclusion
bodies and renaturation were needed.
Key
words:
VEGF, sarkosyl, solubilization, purification, GST. |
