106 results about "Free protein" patented technology

This invention provides an in vitro cell-free protein synthesis system, its reagent kit, and its applications, belonging to the field of protein synthesis technology. This invention improves an in vitro cell-free protein synthesis system based on Kluyveromyces lactis by adding exogenous L-arabinose, significantly enhancing the system's protein synthesis capacity. Furthermore, it provides a more efficient and higher-throughput in vitro protein synthesis reagent kit and a method for synthesizing exogenous proteins. The improvements provided by this invention can be achieved without molecular modification, are simple and convenient to operate, and save costs.

The invention relates to an extracellular vesicle surface protein detection paper-based kit and application thereof, and belongs to the technical field of biology. The technical problems that an existing sEVs detection technology is insufficient in specificity, depends on professional instruments and cannot meet the instant detection requirement are solved. According to the method, sEVs are synergistically captured through double aptamers, the target specificity is improved, signals are amplified in a cascaded mode through hyper-branched rolling circle amplification (HRCA) and a CRISPR / Cas12a system, and visual detection is achieved by detecting FAM-biotin probe separation before and after cutting through a transverse flow test strip. According to the method, interference of free protein is effectively avoided through double-target recognition, and the detection limit is as low as 406 parts / mL; cRISPR and a paper-based technology are combined to realize high sensitivity and instant detection; the system is simple and convenient to operate, does not need professional instruments, is suitable for tumor diagnosis and treatment monitoring of clinical samples, and provides a portable solution for precise medical treatment.

The invention provides an ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting an anti-MDA5 antibody and a preparation method of the ELISA kit. According to the kit, full-length human MDA5 protein (1025 amino acids) expressed by a cell-free protein expression system under the low-temperature condition of 22 DEG C is adopted as a coating antigen, and a disulfide bond enhancer E6820 is added into the system to promote correct folding of the protein. The kit comprises a microwell plate coated with a full-length MDA5 antigen, an enzyme-labeled anti-human IgG secondary antibody, a standard substance with the concentration range of 1-243 ng / mL, a chromogenic substrate and an optimized confining liquid system. The invention solves the problem of insufficient sensitivity caused by the use of fragment antigens in the prior art, and can be used for early diagnosis and illness monitoring of dermatomyositis-related interstitial lung diseases.

The application provides an in-vitro cell-free protein synthesis system and kit containing exogenous magnesium ions and application thereof, including a D2P system (DNA-to-Protein system) and an mR2P system (mRNA-to-Protein system), and belongs to the technical field of protein synthesis. The in-vitro cell-free protein synthesis system containing exogenous magnesium ions adopts magnesium aspartate as a novel magnesium ion source, and especially in a eukaryotic cell-free system, compared with a traditional magnesium ion source, can significantly improve the protein synthesis efficiency and protein expression amount. A more efficient and higher-throughput in-vitro protein synthesis kit and a synthesis method of exogenous proteins are also provided, and the kit has the advantages of simplicity, convenience and low cost.

96 New PCT-Patent Application based on EP 24 186 635.9 Insempra GmbH Vossius Ref.: AG4141 PCT S3 Abstract The present invention relates to a linear double-stranded deoxyribonucleic acid (dsDNA) molecule comprising one or more Tus protein (Tus) binding site(s) at the 5'-terminus, one or more Lac repressor protein (LacI) binding site(s) at the 3'-terminus, and a segment comprising a DNA sequence of interest (DOI) between said Tus binding site(s) and said LacI binding site(s). The present invention further relates to a non-naturally or naturally occurring RNA molecule as encoded by or transcribable / transcribed from the linear dsDNA molecule of the invention, and to an expression DNA cassette comprising a promoter, an RBS, a GOI encoding a POI, and one or more LacI binding site(s), wherein said expression cassette does not comprise a terminator sequence between the 3´ end of said GOI and said one or more LacI binding site(s). The present invention also relates to a non-naturally or naturally occurring RNA molecule as encoded by or transcribable / transcribed from said expression cassette. The present invention further relates to method of protecting a linear deoxyribonucleic acid (DNA) molecule from exonuclease degradation by adding one or more Tus binding site(s) at the first terminus of the DNA molecule and adding one or more LacI binding site(s) at the other terminus of the DNA molecule. The present invention further relates to a method of synthesizing a protein of interest (POI) in a cell- free protein synthesis (CFPS) reaction mixture by using the (ds)DNA and / or RNA molecules of the invention, and also to a cell-free biological system or CFPS reaction mixture comprising the (ds)DNA, expression cassette and / or RNA molecules of the invention.

The invention relates to the technical field of bioengineering, in particular to a preparation method and application of an antibacterial peptide, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID No: 1 and is named as L8. The preparation method of the antibacterial peptide L8 comprises the following steps: S1, obtaining an L8 target gene segment; connecting an L8 target gene segment with a pLC vector to obtain a connection product; transforming the connection product into an escherichia coli DH5-alpha competent cell, and screening positive clones to obtain a pLC-L8 recombinant plasmid; s2, taking the pLC-L8 recombinant plasmid as a substrate, and carrying out cell-free protein synthesis reaction to obtain the antibacterial peptide L8, the cell-free protein synthesis system comprises 175 mM of potassium glutamate, 10 mM of ammonium glutamate, 2.7 mM of potassium oxalate, 33 mM of PEP, 38 mM of compound amino acid, 10 mM of magnesium glutamate, 2% (w / v) PEG-8000, 5 nM of pLC-L8 recombinant plasmid, 11.25 mM of lactose, 33.33% (v / v) of crude extract, 100 mg / mL of escherichia coli tRNA and 2.5 nM of T7 RNA polymerase. The antibacterial peptide L8 is obtained by optimizing the antibacterial peptide LRGG, and compared with the LRGG, the antibacterial peptide L8 subjected to cell-free protein synthesis reaction expression has better antibacterial activity and biological safety.

The invention discloses base-modified mRNA (messenger Ribonucleic Acid) and application thereof in a cell-free protein synthesis system, and belongs to the technical field of nucleic acid modification. Natural nucleoside triphosphate is completely replaced by different types of basic group modified nucleoside triphosphate, mRNA with different chemical modifications is obtained through an in-vitro transcription method and purification, and the mRNA is put into an in-vitro cell-free protein synthesis system for reaction. A reaction element recombination system-PURE system based on prokaryote Escherichia coli purified protein and an eukaryote CFPS system taking rabbit reticulated red lysate as a material are mainly selected to detect the corresponding translation efficiency of mRNA modified by different bases in two cell-free protein synthesis systems respectively. Different regulatory activities may be shown in different cell-free protein synthesis systems when different modified nucleotides are doped into mRNA, base modified triphosphoric acid capable of improving the translation efficiency of an in-vitro protein synthesis system is screened out, and the stability of mRNA in the cell-free protein synthesis system and the corresponding protein expression level are improved.

The invention provides an in-vitro cell-free synthesis method and kit of foreign protein, and belongs to the technical field of protein synthesis. According to the in-vitro cell-free synthesis method of the foreign protein, a water-phase in-vitro cell-free protein synthesis system and a nucleic acid template for coding the foreign protein are provided, a hydrophobic interface in surface contact with the water-phase in-vitro cell-free protein synthesis system is provided, incubation reaction is carried out, the foreign protein is synthesized, and the in-vitro cell-free synthesis method of the foreign protein is obtained. The protein synthesis efficiency and the protein expression quantity of the cell-free system are improved. The invention further provides an in-vitro protein synthesis kit which is more efficient and higher in throughput. The invention provides a simple and convenient hydrophobic interface optimization method which is simple and convenient to operate and saves cost.

The object of the present invention is to provide a protein production method capable of producing an active protein with high efficiency even at a low temperature, and a cell-free protein synthesis kit. A protein production method including producing a protein with a reaction solution of a cell-free protein synthesis system containing either one or both of a cold shock protein and a nucleic acid containing a coding region encoding an amino acid sequence of the cold shock protein. A cell-free protein synthesis kit including one or both of a cold shock protein and a nucleic acid containing a coding region encoding an amino acid sequence of the cold shock protein, and a reaction solution of a cell-free protein synthesis system.

The invention provides a method for inserting an unnatural amino acid and application thereof, the method adopts a pyrrolysine aminoacyl-tRNA synthetase mutant as an orthogonal translation element to introduce the unnatural amino acid into a protein to obtain a protein containing the unnatural amino acid, the invention also provides an in-vitro cell-free protein synthesis system for inserting the non-natural amino acid, and the system can efficiently introduce the non-natural amino acid, especially the lysine analogue non-natural amino acid. The technical bottleneck that a natural translation system is low in non-natural amino acid recognition efficiency is solved, and an efficient and controllable technical tool is provided for protein function research and biological medicine development.

The application discloses a kind of bionic transmembrane protein affinity chromatography column and its preparation method and application.The system (iSTAC) realizes the in-situ construction of transmembrane protein in highly bionic dynamic microenvironment by integrating cell-free protein synthesis, functional mesoporous silica modification and amphiphilic (AH) peptide stabilized planar lipid bilayer technology.The core is to use long-chain PEG-24 crosslinking agent to retain lipid bilayer on the surface of silica gel, provide sufficient conformational dynamic space for multi-transmembrane protein, and introduce AH peptide to repair membrane defects, ensure that the receptor realizes directional embedding while maintaining natural functional attributes.The preparation cycle is shortened from 168 hours to 5 hours, which significantly improves the efficiency of targeted drug screening and in-situ analysis of binding sites.Using this platform, 5-HT 1A Receptor agonists crocin I and crocin II with anti-insomnia and neuroprotective effects are successfully screened from saffron, and the action site is accurately depicted.

Provided herein are methods of cell-free protein synthesis, optimized cell-free protein synthesis (CFPS) reagents, and methods of optimizing CFPS reagents to increase protein expression yield using additional NTPs and / or additional energy sources added with nucleic acid templates. The methods are suitable for protein expression on microfluidic devices having hydrophobic surfaces by combining droplets on the device for parallel screening of a series of expression compositions.

The application provides a SUMO tag-based cell-free protein synthesis system and method. By inserting a SUMO tag at one end of a target protein, and using a cell-free protein expression system containing a protease capable of cutting the SUMO tag, the yield and efficiency of protein synthesis can be improved, the process is shortened, and the cost is reduced.

The utility model relates to the technical field of biomedical instruments, in particular to a cell-free protein synthesis reactor which comprises an outer sleeve, a protein synthesis tube and a pop-up component, the protein synthesis tube is mounted on the inner side of the outer sleeve, the pop-up component comprises a mounting ring, an abutting plate, a driving component and a driving plate, the mounting ring is fixedly connected with the protein synthesis tube, and the abutting plate is fixedly connected with the driving component. The mounting ring is fixedly connected with the outer sleeve and located on the side, away from the outer sleeve, of the protein synthesis tube, the abutting plate is fixedly connected with the mounting ring and located on the side, away from the protein synthesis tube, of the mounting ring, the driving plate is connected with the outer sleeve through the driving component, the driving component drives the driving plate to move, and the driving plate abuts against the abutting plate, so that the protein synthesis tube and the outer sleeve are separated more stably and easily; and therefore, the use safety is improved.

Disclosed are methods for detecting a target molecule in a test sample using a cell-free protein synthesis (CFPS) reaction. The methods may be utilized for detecting target molecules which may include small molecules and / or metabolites of small molecules. The methods employ one or more transcription templates that encode and conditionally express one or more exogenous RNA polymerases in the presence of the target molecule. The expressed RNA polymerases in turn induce expression of one or more reporter molecules from transcription templates comprising promoters for the RNA polymerases, thereby amplifying an output signal that is generated in the presence of a detected target molecule.

Disclosed are compositions, formulations, systems, and methods for cell-free protein synthesis, which comprise a unique combination of cell-free metabolites and non- phosphorylated energy substrates to enable protein synthesis at 2-10x greater yields than alternative systems using non-phosphorylated energy substrates.

Method for continuous cell-free protein synthesis using a system with an inner compartment where the synthesis takes place and an outer compartment both having separate reservoir and supply lines and both being separated by a membrane (tubular) having a cut off of 30kDa so that expressed protein can exit the inner compartment to the outer compartment for any assembly, modification or purification or for bacteriophage formation when the phage RNA is also in the outer compartment.

The invention discloses a high-yield cell-free protein synthesis system based on a cell crude extract and application of the high-yield cell-free protein synthesis system. The system comprises an escherichia coli extract, a DNA template, a buffer solution, an amino acid mixture, a nucleoside triphosphate mixture, an ATP regeneration system and antibiotics, the antibiotics are selected from compounds having inhibitory activity on metabolic processes except ribosome functions in prokaryotic microorganisms, including but not limited to one or more combinations of interfering DNA replication, DNA transcription, cell wall synthesis, energy metabolism, lipid synthesis or other non-translational related metabolic pathways. According to the method disclosed by the invention, the escherichia coli crude extract is used as a bacterial chassis, and non-essential metabolic pathways in a CFPS system are selectively inhibited by introducing different types of antibiotics, so that the yield of protein synthesis is increased.

Provided herein are methods of cell-free protein synthesis, optimised cell-free protein synthesis (CFPS) reagents, and methods for optimising CFPS reagents to increase protein expression yields via high throughput screening of a selection of expression compositions in parallel using low volumes of reagents. The methods comprise the steps of adding enzymes for CFPS to a plurality of isolated volumes, wherein the isolated volumes are covered with a hydrophobic fluid, and adding a nucleic acid template in aqueous solution to the plurality of isolated volumes such that the aqueous layer sinks through the hydrophobic fluid layer and mixes with the previously provided enzyme composition. The methods are applicable to protein expression in tubes or plates using a hydrophobic fluid layer to prevent evaporation.

The invention provided herein relates to methods for measuring protein aggregation and stability. The method can be used for characterizing cell-free protein synthesis reactions.

The invention provided herein relates to methods for normalizing the concentration of beads in droplets on microfluidic devices, as well as methods for cell-free protein synthesis and characterization of expressed proteins.

Provided is a serum-free protein-free medium for culturing mycoplasma, the medium comprising: (a) a basal medium; (b) a lipid mixture containing cholesterol, palmitic acid and oleic acid; (c) a polyvinyl alcohol-based resin; and (d) cyclodextrin.

The present invention relates to a method of synthesizing a (poly)peptide or protein of interest (POI) in a cell-free protein synthesis (CFPS) reaction mixture. The present invention further relates to a cell-free biological system which is or comprises a CFPS reaction mixture as defined or used in accordance with the method of the invention. The present invention also relates to the respective CFPS reaction mixture, and CFPS buffer / buffer composition.

The application discloses an anti-interference reagent and application thereof, and relates to the technical field of biological detection. The application provides the reagent which can reduce the interference in the immunodetection sample in the presence of the interference encountered in the existing antigen-antibody sandwich reaction. The reagent comprises 0.1-1 parts of a blocking agent, 10-25 parts of a non-ionic surfactant and 5-20 parts of fat-free protein. The reagent can be directly added into a detection system, can effectively avoid or reduce the false results in the sample test, and can maintain the accuracy and stability of the detection results.

This specification discloses a food product containing animal-free protein and a method for producing the food product, wherein the animal-free protein can be a protein that is not obtained from animals, or a fragment or cleavage product of a protein.

The present invention provides methods and systems for determining the probability that a protein may be synthesized in a cell-free system. The invention provides a system that receives data comprising the properties of a protein to be synthesized, connects one or more databases comprising protein synthesis data, and calculates the probability of success for the protein to be synthesized. The inventions provide a prediction on whether a protein can or cannot be made. The system leverages protein databases to provide data on how proteins are made based on their digital sequence. The invention reduces unnecessary experimentation, synthesis costs, and further identifies success parameters to reduce the number of actions taken to get demonstrable synthesis.WO

The invention relates to the field of biotechnology and molecular detection, and particularly provides a conjugate and application thereof, the conjugate comprises a solid phase carrier; the substance is coupled with the solid phase carrier and specifically binds to the p24 protein. The conjugate provided by the invention can accurately evaluate the p24 protein content of lentivirus particles without being influenced by the content of free p24 protein in a solution, so that an accurate lentivirus titer result is obtained, and the process stability and accuracy in the lentivirus production process are improved.

Disclosed are methods, compositions, systems, and protein compounds for the directed evolution of enzymes and proteins. The method comprising generating variant protein with a desired functionality, comprising one or more DNA expression templates comprising nucleic acid sequences encoding a variant protein, expressing the variant protein using cell-free protein synthesis; and analyzing one or more parameters associated with the variant protein.

Obtained is: a method for efficiently producing a polypeptide containing a non-canonical amino acid; a method for incorporating a non-canonical amino acid into a polypeptide; a method for producing tRNA bound to a non-canonical amino acid; a non-canonical amino acid incorporation system; a material for use in these methods; or the like. Used is a method for producing a polypeptide containing a non-canonical amino acid, comprising: a step of contacting PylRS from an organism belonging to the order Methanomassiliicoccales or Thermoplasmatales with the non-canonical amino acid; and an incorporation step selected from step (a) of incorporating the non-canonical amino acid into the polypeptide with higher efficiency than in a case of using a cell-free protein synthesis system with PylRS of Methanosarcina mazei (MmPylRS) or step (b) of incorporating the non-canonical amino acid into the polypeptide with higher efficiency than in a case of using an Escherichia coli protein synthesis system with a vector carrying a gene for the PylRS under regulation by a glmS promoter or a case of using an Escherichia coli protein synthesis system with a vector carrying a gene for the PylRS under regulation by a glnS promoter. Alternatively, used is a non-canonical amino acid incorporation system containing highly concentrated PylRS from an organism belonging to the order Methanomassiliicoccales or Thermoplasmatales.

Adhesive formulations comprising bovine serum albumin, b-lactoglobulin, and / or a-lactalbumin can be applied and cured underwater to form a strong adhesive. Curing can be accomplished using heat or via a chemical denaturing component.