Other

Cellfree Sciences Co., Ltd.

Tomio Ogasawara

Yaeta Endo

Tatsuya Sawasaki

Shoichiro Ozaki

Tomonori Hoshiko

Methods to construct a transcription template for cell-free protein synthesis that has a high translation template activity, using a 3'-side primer and a 5&#x2032;-side primer for PCR are provided. The 5&#x2032;-side primer for PCR has a sequence complementary to a base sequence containing at least a part of a promoter functional site from the 5&#x2032;-end of a promoter and has a base sequence that does not contain a base sequence complementary to at least a part of a RNA polymerase-recognizing site of the 3&#x2032;-end of the promoter. The other primer has a base sequence complementary to at least a part of the RNA polymerase-recognizing site of the 3&#x2032;-end of the promoter and has a sequence that does not contain a base sequence complementary to at least a part of a promoter functional site from the 5&#x2032;-end of the promoter.

Transcription template for cell-free protein synthesis and method using the same

One embodiment of the present invention is a diffusion continuous batch cell-free protein-synthesis method characterized simultaneously by continuously supplying substrate and energy source molecules in the supply phase to the reaction phase by the free diffusion via interface between both phases and by transferring by-products formed in the reaction phase by enhancing the efficiency of the synthesis reaction by prolonging the reaction lifetime by directly contacting a synthesis reaction mixture (reaction phase) containing a biological extract with a substrate- and energy source-supplying solution (supply phase) without using barrier such as semi-permeable membrane or ultrafiltration membrane in a general cell-free protein-synthesis reaction means. Another embodiment of the present invention is a dilution batch cell-free protein synthesis method characterized by enhancing the efficiency of the protein synthesis by prolonging the reaction lifetime by adding a diluting solution to the reaction mixture after pre-incubating the reaction mixture in a cell-free protein-synthesis reaction means using a wheat-embryo extract. Another embodiment of the present invention is a method characterized by enhancing the efficiency of the synthesis reaction simultaneously by re-supplying substrate and energy sources necessary for the protein synthesis (e.g., amino acids, ATP, GTP, creatine phosphate) to the reaction mixture using a gel filtration column and/or semipermeable membrane and by discontinuously removing by-products formed during the reaction after the synthesis reaction stops in the batch cell-free protein synthesis method.

Methods of synthesizing cell-free protein

A 1-(N-substituted carbamoyl)-5-fluorouracil derivative represented by the following general formula: ##STR1## R is a 4-alkoxycyclohexyl group-containing radical which is 2,3,4-trimethoxycyclohexcylethyl, 2,3,4-trimethoxycyclohexyl, 2,4-dimethoxycyclohexylmethyl, 3,4-dimethoxycyclohexylethyl, 2,4-dimethoxycyclohexyl, 2,4,5-trimethoxycyclohexyl, 3,4-dimethoxycyclohexylmethyl, 3,4,5-trimethoxycyclohexyl, 4-ethoxycyclohexyl or 4-ethoxycyclohexylmethyl. The compounds are carcinostatic agents. The compounds are used in a therapeutically effective amount in the presence of a carrier.

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Iyo, Japan

Tomio Ogasawara