One Hundred Seventy-Fold Increase in Excretion of an FV Fragment- Tumor Necrosis Factor Alpha Fusion Protein (sFV/TNF-a) from Escherichia coli Caused by the Synergistic Effects of Glycine and Triton X-100

To target tumor necrosis factor alpha (TNF-a) to tumor cells, recombinant DNA techniques were used to construct and express the fused gene VKLVH–TNF-a, which encodes the secreted form of single-chain fusion protein sFV/TNF-a in Escherichia coli. sFV/TNF-a 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 mmol of isopropyl-b-D-thiogalactopyranoside (IPTG) per liter was 16and 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-a 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-a in E. coli by using glycine and Triton X-100.