Cellfree Sciences Co., Ltd.

Other

Yokohama City University

Ehime University

Yaeta Endo

Tatsuya Sawasaki

Shigemichi Nishikawa

Tomio Ogasawara

Yoshihisa Kawamura

Bunsei Kawakami

Toshihiro Kuroita

Ichiro Aoki

Tomoaki Ishigami

Yuko Matsubara

Takayasu Kawasaki

Penmetcha Kumar

Provided are a detection method for a myriad of proteins involved in an autoimmune disease with high sensitivity and high efficiency, and an analysis method for data resulting from the detection method. In order to construct the detection method and analysis method, there is provided means for comprehensively analyzing the proteins involved in an autoimmune disease by bringing a mammal-derived protein expressed in a cell-free protein synthesis system into contact with a sample derived from a patient with an autoimmune disease to detect autoantibody production, and subjecting the detected data to statistical analysis processing, and further, gene ontology analysis and/or pathway analysis.

Method for analyzing proteins contributing to autoimmune diseases, and method for testing for said diseases

The present invention provides a method for preparing cell extract for high-performance cell-free protein synthesis reactions, comprising the elimination of low molecular weight substances that have protein synthesis inhibitory activity, from cell extracts that have cell-free protein synthesis activity, by such methods as dialysis, gel filtration and ultrafiltration. Also provided is a ready-made cell-extract for cell-free protein synthesis employing this method. Furthermore, the formation of insoluble matter can be reduced by performing the process for eliminating the low molecular weight substances that inhibit protein synthesis in the co-presence of ATP, GTP or amino acids, so as to stabilize the cell extract.

Cell extract for cell-free protein synthesis and process for producing the same

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

The present invention presents construction of a detection method requiring no step of removing free biotin during preparation of a biotinylated protein having a biotin tag, in a detection method of a substance interacting with a protein, and studied various preparation methods of the biotinylated protein. In order to solve the above-mentioned problem, the present inventor has found that in a cell-free protein synthesizing system, in particular, a wheat embryo cell-free protein synthesizing system, when biotinylation is performed during or after protein's synthesis, the biotinylation of the protein can be attained in an remarkably lower concentration of the biotin than that in the conventional biotinylation operations, and has accomplished the present invention by using the protein having the biotin tag in each detection system.

Preparation method of biotinylated protein and detection method using the same

The present invention is intended to provide a safe and quick means for screening a drug to a bioactive protein, in particular, an inhibitor, using a cell-free protein synthesis system with the use of a wheat embryo extract solution. The present inventors have strenuously studied to solve the matters above and finally completed the present invention by, in a system with the use of a wheat embryo, among cell-free protein synthesis means, constructing a synthesis system of a bioactive protein while sustaining its activities, and constructing a system for screening an inhibitor candidate to SARS 3CL, as an example using the synthesis system.

High throughput screening method of drug for physiologically active protein

The present invention provides a cell-free protein synthesis method characterized by a cell-free protein synthesis reaction solution that is weakly reducing and suitable for protein folding, performing a weakly reducing synthesis reaction, and preferably performing a translation reaction with the further addition of a substance catalyzing a disulfide bond exchange reaction at the beginning of the translation reaction. This method allows for proper formation of intramolecular disulfide bonds in the protein and efficiently provides protein having substantially the same function as the original protein. Specifically, it provides antibody protein that binds specifically to an antigen.

Reaction solution for cell-free protein synthesis, method of preparing the same, and protein synthesis method using the same

Disclosed are a preparation containing cell extracts for cell-free protein synthesis, prepared by excluding from a living organism a system, participating to inhibiting of self protein synthesis reaction, an apparatus for cell-free protein synthesis reaction equipped with a reaction tank for cell-free protein synthesis, and a kit for use therefor; the preparation can be stored at room temperature and prepared as a preparation in a state where biological functions of the cell extracts are maintained, and further, disclosed is methods for cell-free protein synthesis comprising providing cell extracts from which an inhibitor for self protein synthesis reaction is substantially excluded, having introduced therein treatment selected from supplement, storage, exchange or discharge with respect to an element selected from at least mRNA serving as a template for synthesis reaction, an energy reproduction system enzyme, a substrate, and an energy source.

Preparation containing cell extracts for cell-free protein synthesis and means for synthesizing protein using the preparation

Utilizing a what embryo cell-free protein synthesis system, there are provided a process for the production of selenomethionine-labeled protein, characterized in that, methionine in a wheat embryo extract for a cell-free protein synthesis obtained by a complete removal of endosperm contaminated is changed to selenomethionine and a cell-free protein synthesis is carried out using a reaction solution composition for protein synthesis containing selenomethionine instead of methionine under a batch condition or a dialysis condition and also the said protein produced as such. There are further provided a process for the production of heavy hydrogen-labeled protein using the same means and also the said protein produced as such.

General means of labeling protein by using wheat embryo cell-free protein synthesis system

The present invention provides a composition for cell-free protein synthesis, which is superior in storage stability in a freeze-dried state, more particularly a freeze-dryable or freeze-dried composition for cell-free protein synthesis, which contains a cell extract for cell-free protein synthesis and inositol, and a freeze-dryable or freeze-dried composition for cell-free protein synthesis containing a cell extract for cell-free protein synthesis, and a deliquescent material in a proportion of not more than 0.01 part by weight per part by weight of a protein in the composition; and a composition for cell-free protein synthesis superior in storage stability in a frozen state, more particularly a freezable or frozen composition for cell-free protein synthesis, containing a cell extract for cell-free protein synthesis and polyhydric alcohol.

Composition for cell-free protein synthesis

Disclosed are a preparation containing cell extracts for cell-free protein synthesis, prepared by excluding from a living organism a system, participating to inhibiting of self protein synthesis reaction, an apparatus for cell-free protein synthesis reaction equipped with a reaction tank for cell-free protein synthesis, and a kit for use therefor; the preparation can be stored at room temperature and prepared as a preparation in a state where biological functions of the cell extracts are maintained and further, disclosed is means for cell-free protein synthesis comprising cell extracts from which an inhibitor for self protein synthesis reaction is substantially excluded, having introduced therein treatment selected from supplement, storage, exchange or discharge with respect to an element selected from at least mRNA serving as a template for synthesis reaction, an energy reproduction system enzyme, a substrate, and an energy source.

Preparation containing cell extracts for synthesizing cell-free protein and means for synthesizing cell-free protein

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.

Growing community of inventors

Matsuyama, Japan

Yaeta Endo