Katsuo Noguchi

Improvement of cytotoxicity of tumor necorosis factor (TNF) by increase in basicity of its N-terminal region

Two new recombinant TNFs (named rTNF-Scw1 and -Scw2) with higher basicity than conventional recombinant human TNF-alpha (rTNF-alpha) in the N-terminal region were constructed. Their sequences were constructed based on those of partially purified cytotoxic factors from the culture supernatant of acute monocytic leukemia cells THP-1, which unlike rTNF-alpha are cytotoxic to T24 bladder carcinoma cells in vitro. These new rTNF-Ss showed a broader cytotoxicity to tumor cells than rTNF-alpha. This increase in the basicity of the N-terminal region over that of conventional TNF significantly increased the cytotoxicity on tumor cells in vivo as well as in vitro.

Antitumor activity of a novel chimera tumor necrosis factor (TNF-STH) constructed by connecting rTNF-S with thymosin beta 4 against murine syngeneic tumors

We have shown the in vivo usefulness of a novel chimera tumor necrosis factor (TNF), called rTNF-STH, which was constituted with human thymosin β4 and recombinant human TNF-SAM1β Tumor necrosis was induced by intravenous injection of a smaller amount of rTNF-STH (1 × 103 U/mouse, 0.67μg/mouse) than rTNF-β or rTNF-S (1 × 104 U/mouse, 2.5–5 μg/mouse). Significant antitumor effects of rTNF-STH to Meth A fibrosarcoma, B 16 melanoma, MH134 hepatoma, or Lewis lung carcinoma (3LL) were observed by systemic injection of rTNF-STH at the maximum tolerable dose of 1 × 104 U/mouse (6.7 μg/mouse); this dose did not cause regression of tumors by conventional rTNF-β. rTNF-STH showed a significant prolongation of its half-life in serum. The average calculated half-life of the chimera protein is about 110 min, which is 15 times longer than that of original TNF-SAMI (7.5 min). On the basis of this prolongation of half-life of rTNF-STH and its efficient hemorrhagic necrotic activity, the antitumor effect of rTNF-STH —as compared with that of the known TNF species—is discussed. Findings indicate that use of the chimera protein to alter the N-terminal region of TNF may be a promising approach to obtain molecules that more favorably attack tumors and other diseases than conventional rTNFs.

Purification of recombinant human tumor necrosis factor precursor from Escherichia coli

To study its biological functions, tumor necrosis factor precursor (proTNF) with a molecular size of 26-KDa was obtained as a recombinant protein from Escherichia coli. The recombinant proTNF was successfully accumulated in the insoluble form, corresponding to about 10-15% of total E. coli proteins. Solubilization, gel filtration and anion exchange chromatography were performed under denatured conditions followed by dialysis in phosphate-buffered saline. These processes removed most of the contaminating bacterial proteins, yielding proTNF with a purity of about 70-80%. This recombinant proTNF is expected to be useful for functional studies on activated macrophages with membrane integrated proTNF.

Conjugation to octa‐arginine via disulfide bonds confers solubility to denatured proteins in physiological solution and enables efficient cell internalization

Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa‐arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate‐buffered saline. However, almost all the R8‐conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary‐K1, Cos‐7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8‐conjugated β‐galactosidase and R8‐conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen‐presenting cells and will benefit research and innovation in vaccine design and discovery.