38 results about "Pore Proteins" patented technology

The invention belongs to the technical field of characterization of target samples, and provides PANX3 biopore protein and application of a mutant and single molecule detection thereof. The PANX3 biopore protein is composed of seven identical subunits, the overall channel is funnel-shaped, and the amino acid sequence is shown as SEQ ID NO: 1. After one or more amino acids of the wild type PANX3 biopore protein are mutated into common amino acids except original amino acids, compared with the wild type PANX3 biopore protein, the mutant can be normally expressed and has stable properties, and the current property and the current signal of the mutant are obviously improved.

The invention belongs to the technical field of methods for characterizing target polynucleotides, and provides application of a biological nanopore protein and a mutant thereof in detection. The biological nanopore protein mutant is obtained by mutating wild type PANX1 nanopore protein, the amino acid sequence of the wild type PANX1 nanopore protein is shown as SEQ ID NO: 1, and the mutation means that one or more amino acids of the wild type PANX1 nanopore protein are mutated into common amino acids except original amino acids, or carrying out truncation or truncation substitution on the C region of the wild type PANX1 nanopore protein to obtain the PANX1 nanopore protein mutant. The wild type PANX1 nanopore protein can be normally expressed after mutation, compared with the wild type PANX1 nanopore protein, the biological nanopore protein mutant is stable in property, the C region is truncated, so that the pore diameter of the contraction region of the protein mutant is enlarged, and the pore channel is prevented from being blocked.

The invention provides a nanopore sensor and an application of the nanopore sensor in sequencing. The nanopore sensor comprises a film layer and pore protein inserted in the middle of the film layer to form a pore channel, and when an electric field force is applied across the film layer, the pore channel generates conductivity; wherein the pore protein comprises a pore protein monomer, and the pore protein monomer comprises (a) a protein with an amino acid sequence as shown in SEQ ID NO: 2; or (b) a protein mutant, the amino acid sequence of the protein mutant is subjected to substitution, deletion and / or addition of one or more amino acids at at least one of the following sites of SEQ ID NO: 2: 80, 82, 83 and the like, and the mutant has the function of forming a pore structure through polymerization; or (c) the mutant has at least 70% of identity with the protein, and the mutant has a function of forming a pore structure through polymerization. The invention provides a novel nanopore sensor which can be used for nanopore sequencing and is suitable for the field of single-molecule sequencing.

The invention belongs to the technical field of characterization of target analytes, and provides application of PANX2 protein and a mutant thereof as biopore protein. The bioporous protein is used for detecting one or more of metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic agents, adenosine triphosphate and monosaccharides. After one or more amino acids of the wild type PANX2 protein are mutated into common amino acids except original amino acids, compared with the wild type PANX2 protein, the PANX2 protein mutant can be normally expressed and is stable in property, a current signal is obviously improved during detection, the up-down amplitude of the current signal is longer, and burrs are fewer.

The invention belongs to the technical field of target sample characterization, and provides application of enterococcus pore-forming toxin mutant protein in nanopore detection. The enterococcus pore-forming toxin mutant protein is EPX1 nanopore mutant protein and is obtained by mutating wild type EPX1 nanopore protein, the amino acid sequence of the wild type EPX1 nanopore protein is as shown in SEQ ID NO: 1, and the mutation means that one or more amino acids of the wild type EPX1 nanopore protein are mutated into common amino acids except original amino acids. The EPX1 protein (wild type) is a type of pore-forming toxin protein and widely exists in various living bodies in nature, and a mutant of the EPX1 protein shows excellent pore-forming capacity after being modified (after amino acid mutation). The structural stability, the molecular recognition efficiency and the detection performance of the EPX1 nanopore mutant protein are remarkably improved.

The invention discloses a mutant PHT nanopore protein monomer and application thereof. The mutant PHT nanopore protein monomer comprises a mutant of which the sequence is shown as SEQ ID NO: 2, and the mutant comprises mutation at at least one position of the 181st position, the 185th position, the 219th position and the 233th position of the SEQ ID NO: 2. On the basis of the wild type PHT nanopore protein, the capture and resolution capabilities of a DNA substrate are improved by means of amino acid mutation and combined mutation.

Provided are a nanopore protein complex, a construction method therefor, and a use thereof. The nanopore complex comprises: a nanopore protein and an accessory protein. The nanopore protein comprises a nanopore cavity, and the nanopore cavity is formed by polymerizing a plurality of pore protein monomers. The accessory protein is formed by polymerizing a plurality of accessory protein monomers. The N-terminus of the accessory protein is embedded in the nanopore cavity and forms a continuous channel together with the nanopore cavity. On the basis of the moving direction of an analyte passing through the continuous channel, the continuous channel comprises a first sensing region and a second sensing region which are communicated sequentially, wherein the first sensing region is formed by part of the nanopore protein, and the second sensing region is formed by part or all of the accessory protein. A nanopore protein monomer is selected from a protein having an amino acid sequence as shown in SEQ ID NO: 1 and a variant thereof, and an accessory protein monomer is selected from a protein having an amino acid sequence as shown in SEQ ID NO: 3 and a variant thereof.

This invention belongs to the field of target characterization technology, and provides an application of PANX2 protein and its mutants as bioporins. The bioporins can detect one or more of the following: metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic reagents, adenosine triphosphate (ATP), and monosaccharides. In this invention, one or more amino acids of the wild-type PANX2 protein are mutated to common amino acids other than the original amino acids. Compared to the wild-type PANX2 protein, the PANX2 protein mutants can be expressed normally, are stable, and show a significantly improved current signal during detection, with a longer amplitude and fewer spikes.

The invention provides a pore protein monomer, a pore protein, a mutant of the pore protein and application of the pore protein monomer and the pore protein. The porous protein monomer comprises: (a) a protein composed of an amino acid sequence represented by SEQ ID NO: 1; or (b) a protein mutant, the amino acid sequence of the protein mutant is subjected to substitution, deletion and / or addition of one or more amino acids at at least one of the following sites of the amino acid sequence shown in SEQ ID NO: 1: 63, 64 and the like, and the protein mutant has the function of forming a pore structure through polymerization; or (c) a pore protein monomer which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity with the protein in (a) or (b) and has a function of forming a pore structure through polymerization. The problem of poor pore stability of mesoporous protein in the prior art can be solved, and the method is suitable for the field of single-molecule sequencing.

A fermented milk product, wherein the fermented milk product comprises:a porous protein network;a first, capsular, preferably negatively charged, exopolysaccharide; anda second, non-capsular, preferably neutral, exopolysaccharide.A starter culture comprising a first, preferably negatively charged, capsular, exopolysaccharide producing lactic acid bacterial strain and a second, preferably neutral, non-capsular, exopolysaccharide producing lactic acid bacterial strain, wherein the weight ratio of the weight of the first exopolysaccharide producing lactic acid bacterial strain to the weight of the second exopolysaccharide producing lactic acid bacterial stain in the starter culture lies in the range from equal to or more than 1:1 to equal to or less than 100:1, more preferably in the range from equal to or more than 10:1 to equal to or less than 100:1; and a use thereof and process using such.

The invention provides a fusion method of pore protein and a membrane, a pore protein jack buffer solution and application. The fusion method comprises the following steps: distributing a first solution on the first side of the membrane, and distributing a second solution on the second side of the membrane; applying voltage to two sides of the membrane, fusing the pore protein and the membrane, and detecting current in the solution; when the current is increased, the pore protein is inserted into the membrane, the voltage is adjusted to 0V, and the fusion of the pore protein and the membrane is completed; the first solution comprises a first buffer solution containing pore protein, and the second solution comprises a second buffer solution without pore protein; the osmotic pressure of the first solution is greater than that of the second solution. The porous protein and membrane fusion single porosity problem in the prior art can be solved, and the method is suitable for the field of biological analysis and detection.

The present invention provides a nanopore protein complex and a use thereof. The nanopore protein complex comprises: a nanopore protein and multiple auxiliary proteins, the nanopore protein comprising multiple porin monomers and a nanopore channel structure formed by polymerization of the porin monomers, each auxiliary protein having an N-terminus thereof located within the nanopore channel structure, and the multiple auxiliary proteins being connected one-to-one with the multiple porin monomers and, together with the nanopore channel structure forming a continuous channel; along a direction in which an analyte passes through the continuous channel, the continuous channel comprises a first constriction region and a second constriction region that are connected in sequence, the first constriction region being formed at least in part by the nanopore protein, the second constriction region being formed in part or in whole by the auxiliary proteins, and the nanopore protein being wild-type or a mutant.

The invention provides a nanopore protein and a modified nanopore protein, and relates to the technical field of biology. The modified nanopore protein provided by the invention can be used as a wide-aperture nanopore, the length of a hydrophobic region of the provided nanopore protein is as long as 35 angstroms, the length of the hydrophobic region is obviously higher than that of the existing protein, and the integrated assembly effect of the protein on lipid bilayers and artificial membranes is enhanced; the modified mutant provided by the invention is beneficial for biomolecules, compounds and ions to selectively enter a channel under the action of electrophoretic force and electroosmosis force, so that selective transportation of the biomolecules, the compounds and the ions is realized; and the modified nanopore shrinkage region is also beneficial to selective interaction of biomolecules, compounds and ions with the channel, so that selective transportation and recognition of the biomolecules, the compounds and the ions are realized, and the method is suitable for application scenes such as molecular detection, cell communication or molecular separation and the like.

The application discloses application of a porin PorA in improving the ammonium nitrogen utilization capacity of a methanotrophic bacterium. The porin PorA has an amino acid sequence as shown in SEQ ID NO. 1. The coding gene porA of the porin PorA has a nucleotide sequence as shown in SEQ ID NO. 2. The application of the porin PorA in improving the ammonium nitrogen utilization capacity of Methylotuvimicrobium buryatense. Inactivation of the porin PorA can make the methanotrophic bacterium Methylotuvimicrobium buryatense grow by utilizing ammonium nitrogen, reduce the cost of fermenting the bacterium, and is of great significance for industrial application of the bacterium.

The application belongs to the technical field of methods for characterizing target polynucleotides, and provides an application of a biological nanopore protein and a mutant thereof in detection. The biological nanopore protein mutant is obtained by mutating a wild-type PANX1 nanopore protein, and an amino acid sequence of the wild-type PANX1 nanopore protein is shown as SEQ ID NO:1. The mutation refers to that one or more amino acids of the wild-type PANX1 nanopore protein are mutated into common amino acids other than original amino acids, or a C region of the wild-type PANX1 nanopore protein is truncated or truncated and replaced to obtain the PANX1 nanopore protein mutant. After the wild-type PANX1 nanopore protein is mutated, the wild-type PANX1 nanopore protein can be normally expressed. Compared with the wild-type PANX1 nanopore protein, the biological nanopore protein mutant is stable in property, and the C region is truncated to expand the pore diameter of the contraction region of the protein mutant, so that the pore channel is avoided from being blocked.

The invention provides a pore protein monomer, a pore protein, a mutant of the pore protein and application of the pore protein monomer and the pore protein. The porous protein monomer comprises (a) a protein with an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein mutant, the amino acid sequence of the protein mutant is subjected to substitution, deletion and / or addition of one or more amino acids at at least one of the following sites in SEQ ID NO: 1: 77, 81, 82 and the like, and the mutant has the function of forming a pore structure through polymerization; or (c) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the protein in (a) or (b), and the mutant has the function of forming a pore structure through polymerization. The problem that in the prior art, nanopore protein is low in library capture rate can be solved, and the method is suitable for the field of single-molecule sequencing.

The invention provides a novel pore protein BCP34 as well as a mutant and application thereof. Specifically, the wild type pore protein BCP34 is a nonaglymer protein, and the amino acid sequence of a monomer of the wild type pore protein BCP34 is SEQ ID NO: 1; the mutant of the pore protein BCP34 monomer comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity with SEQ ID NO: 1, and an amino acid sequence having mutation at one or more positions in a sensor region, a transmembrane region, an inlet, a barrel inner wall and an outlet of a wild type pore protein BCP34 monomer. The wild type pore protein BCP34 provided by the invention has the capability of being applied to nanopore sequencing, and the pore protein BCP34 mutant obtained by optimization has higher library capture capability and nanopore sequencing accuracy.

The present invention discloses a nanopore sequencing method mediated by liposome vesicles, which relates to the field of genomic sequencing. First, a closed circuit is formed in two half-pools within a flow cell. Then, multiple vesicles formed by liposomes are slowly pipetted into the organic phase using a syringe. The inner pore protein of the syringe and the sequencing sample are encapsulated in each vesicle. After the vesicles are driven by an electric field to float near the channel pore, they are punctured, and the single lipid molecular layer around the channel becomes a bilayer lipid membrane. The translocation voltage is controlled to insert the pore protein into the bilayer lipid membrane to form a nanopore in the middle of the two half-pools. A speed control protein is added near the nanopore, thereby driving the nucleic acid of the sequencing sample near the nanopore to continuously pass through the pore for sequencing at a set translocation speed. The method of the present invention can achieve a relatively high sequencing speed by regulating the speed of the protein, and the nanopore sequencing process is stable, with simple operation, high efficiency, and can achieve a relatively high capture rate.

The present invention belongs to the technical field of characterization of target analyte properties, and particularly provides a mutant of a porin monomer, a protein pore comprising same, and use thereof in the detection of a target analyte, wherein an amino acid of the mutant of the porin monomer comprises a sequence set forth in SEQ ID NO: 1 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, or 50% identity thereto, and the amino acid of the mutant of the porin monomer comprises mutations at one or more positions corresponding to T71, S75, or F76 of SEQ ID NO: 1.

The invention relates to peptides and compounds that bind a two partner secretion (TPS) system protein. In some embodiments of the invention, the peptide or compound binds to a translocator pore protein (TpsB) thereby reducing translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane.

The application belongs to the technical field of biochemistry and molecular biology, and provides application of a two-component toxin nanopore protein XaxAB after site-directed mutation modification. The two-component toxin nanopore protein XaxAB comprises equal numbers of XaxA subunits and XaxB subunits, the amino acid sequence of the XaxA subunit is shown as SEQ ID NO: 1, and the amino acid sequence of the XaxB subunit is shown as SEQ ID NO: 2. The two-component toxin nanopore protein XaxAB is subjected to site-directed mutation modification, T290C mutation is introduced into the XaxA subunit and I30C mutation is introduced into the XaxB subunit, the two-component toxin nanopore protein XaxAB after site-directed mutation modification improves the current signal quality of pore opening, sequencing or detection, the current property is stable, the current signal width is narrower, and burrs are less.

The application belongs to the technical field of characterizing target samples, and provides application of enterococcus pore-forming toxin mutant protein in nanopore detection. The enterococcus pore-forming toxin mutant protein is an EPX1 nanopore mutant protein, which is obtained by mutation of wild-type EPX1 nanopore protein, and the amino acid sequence of the wild-type EPX1 nanopore protein is shown as SEQ ID NO: 1. The mutation refers to that one or more amino acids of the wild-type EPX1 nanopore protein are mutated into common amino acids other than the original amino acids. The EPX1 protein (wild type) of the application is a kind of pore-forming toxin protein, which widely exists in various organisms in nature. The mutant thereof exhibits excellent pore-forming ability after modification (after amino acid mutation). The EPX1 nanopore mutant protein realizes significant improvement in structural stability, molecular recognition efficiency and detection performance.

The present invention relates to novel Puncture protein monomers, Puncture protein pores formed from said monomers and the use of said Puncture protein pores in analyte detection and characterization.

Disclosed in the present invention are a nanopore protein monomer and a use thereof. The nanopore protein monomer is any one of the following polypeptides: (a1) a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 4; (a2) a polypeptide derived from the amino acid sequence as set forth in SEQ ID NO: 4 by means of substitution and / or deletion and / or addition of one or more amino acids and having the same function; (a3) a polypeptide having 80% or above identity to the amino acid sequence defined in any one of (a1)-(a2) and having the same function; and (a4) a fusion polypeptide obtained after linking a tag to the terminus of the polypeptide defined in any one of (a1)-(a3). In the present invention, a nanopore protein which is stable and can be used for DNA detection is obtained on the basis of nanopores without a natural constriction region by a method of artificially designing a contraction region.

The present disclosure relates to relates methods and associated compositions that provide fast, efficient site-selective conjugation of a protein, such as the pore-forming protein α-hemolysin, to a biomolecule, such as a DNA polymerase, and the use of such site-selective protein-biomolecule conjugates in nanopore devices and methods.

The invention discloses a method for embedding nanopore protein into a biomimetic membrane and application of the biomimetic membrane. The method comprises the steps that the biomimetic membrane of a multi-block copolymer is prepared, and the multi-block copolymer contains a hydrophilic segment and a hydrophobic segment; embedding the nanopore protein into the biomimetic membrane; wherein the multi-block copolymer comprises a copolymer with two or more blocks; the biomimetic membrane contains an aromatic ring structure, and the aromatic ring structure is connected between the hydrophilic segment and the hydrophobic segment of the multi-block copolymer; and / or the nanopore protein contains an aromatic ring structure, and the aromatic ring structure is located on the amino acid, facing one side of the biomimetic membrane, of the transmembrane region of the nanopore protein. According to the method, the number of single holes and / or the single hole reserving rate during hole embedding of the biomimetic membrane can be increased.

This application provides a method and application for protecting nanoporins. The protection method includes: embedding the nanoporin onto a membrane, the membrane dividing a solution into two parts, which are connected through the nanoporin; and applying a negative voltage to one side of the solution containing the nanoporin before membrane crosslinking. This application enables an initiator to approach the membrane and undergo membrane crosslinking while remaining away from the nanoporin, thus protecting the nanoporin and preventing damage that could cause abnormalities in the opening current or even subsequent sequencing current.

A novel pore protein suitable for nanopore detection is provided, the pore protein is formed by polymerization of a pore protein monomer, the pore protein monomer comprises a cylinder segment, and the cylinder segment: a, has an amino acid sequence as shown in SEQ ID NO: 1; b, compared with the amino acid sequence as shown in SEQ ID NO: 1, the cylinder segment has an amino acid sequence formed by substitution, deletion and / or addition of one or more amino acids, and the cylinder segment has a function of forming a pore structure through polymerization; or c, compared with the amino acid sequence as shown in SEQ ID NO: 1, the cylinder section has an amino acid sequence with the identity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%, and the cylinder section has the function of forming a pore channel structure through polymerization.

The invention provides a nanopore protein BCP22, which is (a), (b) or (c) as follows: (a) a protein with an amino acid sequence as shown in SEQ ID NO: 1; b) a protein which has more than 70% of identity with the SEQ ID NO: 1 and has the function of the SEQ ID NO: 1; and c) a fusion protein obtained by adding a label at the tail end of the protein sequence shown in the a) or the b), and can be applied to nanopore sequencing and detection aiming at small molecules, DNA, RNA, polypeptides and the like.

The invention discloses a nanopore protein monomer and application thereof. The nanopore protein monomer is any one of the following polypeptides: (a1) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 4; (a2) a polypeptide which is obtained by substitution and / or deletion and / or addition of one or more amino acids on the amino acid sequence as shown in SEQ ID NO: 4 and has the same function as the amino acid sequence as shown in SEQ ID NO: 4; (a3) a polypeptide which has more than 80% of identity with an amino acid sequence defined by any one of (a1)-(a2) and has the same function; and (a4) a fused polypeptide obtained by connecting a tag to the tail end of any polypeptide defined in (a1)-(a3). The stable nanopore protein capable of being used for DNA (deoxyribonucleic acid) detection is obtained by adopting an artificial constriction region design mode on the basis of a natural nanopore without a constriction region.