170-FOLD INCREASE IN EXCRETION OF AN FV FRAGMENT-TUMOR NECROSIS FACTOR-ALPHA FUSION PROTEIN (SFV TNF-ALPHA) FROM ESCHERICHIA-COLI CAUSED BYTHE SYNERGISTIC EFFECTS OF GLYCINE AND TRITON X-100/

To target tumor necrosis factor alpha (TNF-alpha) to tumor cells, reco mbinant DNA techniques were used to construct and express the fused ge ne VKLVH-TNF-alpha, which encodes the secreted form of single-chain fu sion protein sFV/TNF-alpha in Escherichia coli. sFV/TNF-alpha was secr eted into the culture medium and purified by affinity chromatography. The production of the fusion protein in the culture medium under the o ptimal conditions of 30 degrees C and 37 mu mol of isopropyl-beta-D-th iogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than th at under the standard conditions of 37 degrees C and 1 mmol of IPTG pe r 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 mechanis m for enhancement of fusion protein production and excretion by treatm ent with glycine and Triton X-100. This study thus provides a practica l, large-scale method for more efficient production of the heterologou s fusion protein sFV/TNF-alpha in E. coli by using glycine and Triton X-100.