One hundred seventy-fold increase in excretion of an FV fragment-tumor necrosis factor alpha fusion protein (sFV/TNF-alpha) from Escherichia coli caused by the synergistic effects of glycine and triton X-100.
To target tumor necrosis factor alpha (TNF-alpha) to tumor cells, recombinant DNA techniques were used to construct and express the fused gene VKLVH-TNF-alpha, which encodes the secreted form of single-chain fusion protein sFV/TNF-alpha in Escherichia coli. sFV/TNF-alpha 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 degrees C and 37 micromol of isopropyl-beta-D-thiogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than that under the standard conditions of 37 degrees 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-alpha 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-alpha in E. coli by using glycine and Triton X-100.
Departments of Microbiology, Saskatoon Cancer Center, College of Medicine, University of Saskatchewan, Canada.
J
J
Yang
T
T
Moyana
S
S
MacKenzie
Q
Q
Xia
J
J
Xiang
D003470:Culture Media; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D005998:Glycine / Q000494:pharmacology*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D015636:Magnesium Chloride / Q000494:pharmacology; D008854:Microscopy, Electron; D017830:Octoxynol / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
D003470:Culture Media; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D015636:Magnesium Chloride; D007544:Isopropyl Thiogalactoside; D017830:Octoxynol; D001616:beta-Galactosidase; D005998:Glycine