146 results about "Cell-free protein synthesis" patented technology

Various nucleic acid-based matrixes are provided, comprising nucleic acid monomers as building blocks, as well as nucleic acids encoding proteins, so as to produce novel biomaterials. Methods of utilizing such biomaterials include cell-free protein synthesis.

Various nucleic acid-based matrixes are provided, comprising nucleic acid monomers as building blocks, as well as nucleic acids encoding proteins, so as to produce novel biomaterials. Methods of utilizing such biomaterials include delivery of biologically active agents, cell and tissue culture, and cell-free protein synthesis.

The invention provides a theoretical design and technical design of cell-free protein synthesis for DNA replication, transcription and translation coupling, a preparation, a kit and a preparation method. Specifically, with the application of the in-vitro cell-free synthesis system provided by the invention, complex protein can be synthesized, and moreover, DNA and mRNA can be synthesized; and effective, high-throughput and quite convenient protein synthesis can be completed with the use of a DNA template by a minute quantity (nanogram-microgram).

It is intended to provide a process for producing a plant germ extract wherein the step of breaking a plant germ into fine pieces is carried out by impacting or cutting and / or in the presence of an extraction solvent, and a germ extract obtained by this process which is contaminated with little impurities unnecessary or exerting undesirable effects in cell-free protein synthesis. Thus, cell-free protein synthesis can be performed at a high stability and a high efficiency.

The invention provides methods for producing a protein in a cell free protein synthesis system such that the protein does not contain an asparagine (Asn or N) residue at serine (Ser or S) positions. Also provided are compositions and nucleic acid templates for use in the methods described herein.

Compositions and methods are provided for the enhanced in vitro synthesis of protein molecules, by optimizing the metabolism of amino acids present in the reaction mix, preferably all amino acids in the reaction mixture. By performing synthesis with extracts from genetically modified microbial strains that are deficient in multiple amino acid metabolizing enzymes reduces the enzymatic activities responsible for catalyzing these reactions and improves the overall yield of synthesis.

The invention provides a method for improving protein synthesis efficiency in cells. Specifically, the present invention provides an engineering bacterial strain for in-vitro cell-free protein synthesis, a first exogenous gene expression cassette of a first nucleic acid construct expressing a first fusion protein is integrated into a genome of the gene engineering bacterial strain, in the gene engineering bacterial strain, the expression or activity of a KlEXN53 gene (nuclease gene) is lowered. A cell extract (such as a yeast cell extract) derived from the engineering bacterial strain of the invention can significantly increase the stability of nucleic acid without the need for additional manual addition of T7 RNP, and the production efficiency of protein in an in-vitro protein synthesis system can be significantly increase.

The present invention provides a preparation method of a yeast extract solution for cell-free protein synthesis, which solution is easy to prepare and is capable of synthesizing a higher amount of a protein than by conventional yeast extract solutions, the yeast extract solution, a cell-free synthesis method of protein, which uses the yeast extract solution, and a kit for cell-free protein synthesis containing the yeast extract solution. The method of the present invention includes rupturing a yeast cell in a frozen state, and obtaining an extract thereof.

Various nucleic acid-based matrixes are provided, comprising nucleic acid monomers as building blocks, as well as nucleic acids encoding proteins, so as to produce novel biomaterials. Methods of utilizing such biomaterials include cell-free protein synthesis.

The present invention provides a method for monitoring of profile changes of components in a dynamic system such as a cell-free in vitro protein synthesis system by using liquid chromatography (LC) combined with mass spectrometry (MS). In an additional aspect, this invention provides a method for enhancing the yield and / or reproducibility in a cell-free protein synthesis system by modulating the level and / or activity of a protein component that has regulatory effects on the system.

Compositions and methods are provided for the in vitro synthesis of biological molecules in reaction mixtures comprising anti-foam agents. The reaction mix comprising antifoam agent may be a scaled up reaction, e.g. in reaction volume greater than at least about 15 ul. Reactions may be performed in various reactors, as known in the art, which include stirred reactors, bubble-column reactors; and the like.

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 3CLpro, as an example using the synthesis system.

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.

Provided is a method of producing a template DNA used for protein synthesis comprising a step of amplifying a linear double-stranded DNA by polymerase chain reaction (PCR), by using a reaction solution comprising a first double-stranded DNA fragment comprising a sequence coding for a protein or a portion thereof, a second double-stranded DNA fragment comprising a sequence overlapping with the 5′ terminal region of the first DNA fragment, a third double-stranded DNA fragment comprising a sequence overlapping with the 3′ terminal region of the first DNA fragment, a sense primer which anneals with the 5′ terminal region of the second DNA fragment, and an anti-sense primer which anneals with the 3′ terminal region of the third DNA fragment, wherein the second DNA fragment comprises a regulatory sequence for transcription and translation of a gene, and the concentrations of the second DNA fragment and the third DNA fragment in the reaction solution each range from 5 to 2,500 pmol / L. The use of this method enables efficient production of a template DNA for expression and purification of a protein.

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.

The invention discloses a method for expression of fatty acid desaturase by an acellular protein synthesis system, and belongs to the enzyme engineering field. The method uses the wheat germ acellular protein synthesis system for cloning and expression of omega 3 desaturase gene (FADS15) from mortierella alpine ATCC32222 (American type culture collection32222) without the need for preparation of mRNA, the expression amount is high, subsequent purification steps are simple, and the method lays the foundation for the next step of research of membrane protein crystal structures and functions.

The present invention provides a novel extract solution for cell-free protein synthesis, which contains at least an extract derived from a Bombyx mori L. tissue and an exogenous mRNA, which is easy to prepare and which is capable of realizing cell-free protein synthesis that can synthesize glycoprotein, a cell-free protein synthesis method using it, and a method for preparing an extract solution for cell-free protein synthesis, which includes adding an exogenous mRNA to an extract derived from a Bombyx mori L. tissue.

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.

This invention relates to an approach for detection of chain truncating mutations based on the utilization of existing sample collection methods such as FOBT platforms, together with advanced methods for cell-free protein expression. “When further combined with mass spectrometry, the invention provides the ability to simultaneously detect changes in the amino acid sequence of multiple peptides. In some embodiments, DNA is isolated from a patient fecal sample and specific regions of a gene (i.e., for example, a K-ras gene or an APC gene) are PCR amplified using specifically designed primers that allow translation of encoded peptide fragments in a cell-free protein synthesis system. Nascent proteins are affinity purified and their mass is detected by MALDI-TOF which allows identifying low levels of mutations.

Prepared is an extract composition having an improved protein synthetic activity in a cell-free protein synthesis system using a mammalian cultured cell extract. An eukaryotic translation initiation factor and / or translational regulator are added to a cell-free protein synthesis system comprising an extract prepared from cultured mammalian cells and a template mRNA. These factors are one or more selected from the group consisting of eukaryotic translation initiation factors 4E (eIF4E), 2 (eIF2) and 2B (eIF2B), and eukaryotic translational regulator p97.

The present invention provides a wheat germ cell-free protein synthesis system for high level expression of recombination snake venom kininogenase. According to the system, snake venom kininogenase gene of agkistrodon halys pallas is cloned through reverse transcription, wheat germ cell-free expression plasmid pCS<2+> / VK is constructed, a wheat germ extract is prepared, an in vitro transcription and translation system is established, the snake venom kininogenase is expressed and the system is optimized. The N terminal of the exogenous protein expressed by the expression system has the His protein tag, and a Ni column elution method is adopted to carry out purification, such that the subsequent purification of the recombinant protein is substantially simplified, the purification efficiency is high, and the cost is low. The exogenous protein further has biological activity, and provides the foundation for the next clinical study.

Provided is a process for producing a protein with post-translational modification in a cell-free protein synthesis system having a higher protein synthetic activity and glycosylation capability. The process of the present invention comprises preparing a cell extract from cultured cells of an immortalized mammalian cell line that has enhanced protein secreting activity, and adding an mRNA encoding a glycoprotein to the cell extract.

This invention relates to methods of producing a rare amino acid- or non-natural amino acid-containing protein in a cell free protein synthesis system and kits for use in and for accomplishing same. Specifically, the methods comprise the steps of expressing at least one orthogonal suppressor tRNA (o-tRNA) / aminoacyl-tRNA synthetase (aaRS) pair or derivatives thereof specific for incorporation of a rare amino acid- or non-natural amino acid in an E. coli organism; preparing a lysate of said E. coli organism expressing said orthogonal suppressor tRNA (o-tRNA) / aminoacyl-tRNA synthetase (aaRS) pair; and contacting said lysate with a template DNA containing a mutant gene in which at least one amino acid codon at a given site of the protein-encoding gene has been mutated into an amber or ochre mutation and further providing a cognate rare amino acid or non-natural amino acid and other factors necessary for protein synthesis; wherein protein synthesis occurs following said contact to produce a protein containing said at least one rare amino acid or said non-natural amino acid. Kits for use are described, as well.

A method for detecting proteins under mutual interaction which comprises mixing a protein having a label for detection synthesized by a cell-free protein synthesis method with another protein having a modification for separation, or synthesizing a protein having a label for detection and another protein having a modification for separation in a single system by the cell-free protein synthesis method; separating a pair of proteins formed by the interaction between these proteins with the use of the modification for separation as described above; and distinguishing these proteins with the use of the label for detection as described above. A screening method using this method.

Methods and kits are provided for calibrating a cell-free protein synthesis reaction for optimal activity. The method includes the steps of providing an extract competent for cell-free protein synthesis (CFPS); performing cell-free protein synthesis with the extract; measuring a first reaction end-point where in vitro protein synthesis plateaus; measuring a second reaction end-point where Energy Charge of the extract declines to a level in a range from about 0.40 to about 0.80 of Energy Charge of a control extract; and adjusting Energy Charge of the extract to a level in a range from about 0.80 to about 1.0 of the control extract.

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′-side primer for PCR are provided. The 5′-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′-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′-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′-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′-end of the promoter.

According to the present invention, a composition possessing cell-free protein synthesis activity with reduced contaminating lipopolysaccharide, and a method for producing a protein using the same are provided. When ribosome display is performed using the composition and method for protein production of the present invention, the background that is caused by non-specific binding is reduced, so that a nucleic acid that encodes the desired polypeptide can be selected with high accuracy and high efficiency.

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.