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Applied and Environmental Microbiology, August 1998, p. 2869-2874, Vol. 64, No. 8
Departments of
Microbiology1 and
Pathology,2 Saskatoon Cancer Center,
College of Medicine, University of Saskatchewan, and
Plant
Biotechnology Institute, National Research Council of
Canada,3 Saskatoon, Saskatchewan S7N 0W0, Canada
Received 25 March 1998/Accepted 2 June 1998
To target tumor necrosis factor alpha (TNF-
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
One Hundred Seventy-Fold Increase in Excretion of an FV
Fragment-Tumor Necrosis Factor Alpha Fusion Protein (sFV/TNF-
) from
Escherichia coli Caused by the Synergistic Effects of
Glycine and Triton X-100
) to tumor cells,
recombinant DNA techniques were used to construct and express the fused
gene VKLVH-TNF-, which encodes the secreted
form of single-chain fusion protein sFV/TNF- in Escherichia
coli. sFV/TNF- was secreted into the culture medium and
purified by affinity chromatography. The production of the fusion
protein in the culture medium under the optimal conditions of 30°C
and 37 µmol of isopropyl-
-D-thiogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than that under the standard
conditions of 37°C and 1 mmol of IPTG per liter. Fusion protein
excretion into culture medium with 2% glycine, 1% Triton X-100, or
both of these two chemicals was either 14-, 38-, or 170-fold higher,
respectively than that without the two chemicals. The final yield of
sFV/TNF- was estimated to be 50 mg/liter. The loss of integrity of
the cellular membrane may be a potential mechanism for enhancement of
fusion protein production and excretion by treatment with glycine and
Triton X-100. This study thus provides a practical, large-scale method
for more efficient production of the heterologous fusion protein
sFV/TNF- in E. coli by using glycine and
Triton X-100.
*
Corresponding author. Mailing address: Saskatoon Cancer
Center, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada. Phone: (306) 655-2917. Fax: (306) 655-2910.
Applied and Environmental Microbiology, August 1998, p. 2869-2874, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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