356 results about "Amino acid mutation" patented technology

The invention belongs to the technical field of protein engineering, and discloses a Brazilian sweet protein mutant and application thereof. According to the invention, lysine is used for replacing aspartic acid at the 29th site of Brazilian sweet protein, and / or arginine is used for replacing glutamic acid at the 36th site, and / or alanine is used for replacing aspartic acid at the 50th site, amino acid mutation at the sites is carried out to construct a Brazilian sweet protein mutant, and the Brazilian sweet protein mutant is subjected to heterologous expression in pichia pastoris GS115, so that the Brazilian sweet protein mutant is obtained. And carrying out sweetness performance evaluation on the expressed Brazilian sweet protein mutant. The sweetness property of the double mutant Asp29Lys-Asp50Ala protein is improved by 240% compared with the sweetness property of the Brazilian sweet protein before mutation.

The embodiment of the invention provides a tyrosine ammonia lyase mutant and application thereof in synthesis of p-coumaric acid. The mutant is subjected to any one of the following amino acid mutations on an amino acid sequence as shown in SEQ ID NO. 2: F497I, F497I + L248M, F497I + L248M + M4T, and L248M + F497I + M4T + V482L. According to the embodiment of the invention, an amino acid sequence as shown in SEQ ID NO. 2 is taken as a template, a key active site of the template is subjected to mutation transformation, a mutant library is constructed, and a series of mutants with remarkably improved catalytic activity and / or conversion rate are screened; the tyrosine ammonia lyase mutant provided by the embodiment of the invention can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield.

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 embodiment of the invention provides a method for obtaining a tyrosine ammonialyase mutant based on high-throughput screening, the tyrosine ammonialyase mutant and application of the tyrosine ammonialyase mutant. The mutant is subjected to any one of the following amino acid mutations on an amino acid sequence as shown in SEQ ID NO.2: A245G + A247T, and D192V + A245G + A247T. According to the embodiment of the invention, an amino acid sequence as shown in SEQ ID NO.2 is taken as a template (basis), a key active site of the template is subjected to mutation transformation, a mutant library is constructed, and a series of mutants with remarkably improved catalytic activity and / or conversion rate are screened; the tyrosine ammonia lyase mutant provided by the embodiment of the invention can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield, which indicates that the mutant has more significant application prospects in catalysis of L-tyrosine to generate p-coumaric acid.

The invention provides formaldehyde lyase as well as a mutant and application thereof. The amino acid sequence of the formaldehyde lyase mutant is shown as SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and contains at least one of the following sites: S26, W86, N87 or Y87, L109 or M109, L110, H281, Q282 or E282 and A460, and the amino acid sequence is subjected to amino acid mutation and has a formaldehyde lyase activity function. The amino acid sequences of the formaldehyde lyase and the mutants of the formaldehyde lyase have at least 99% of sequence identity with SEQ ID NO.1-7. The formaldehyde lyase and the mutant thereof can catalyze formaldehyde to synthesize 1, 3-dihydroxyacetone, and a new way for converting formaldehyde into 1, 3-dihydroxyacetone is provided.

The invention discloses a Taq DNA polymerase mutant as well as a preparation method and application thereof, and relates to the technical field of biology. The Taq DNA polymerase mutant is mutation on the basis of a wild type Taq DNA polymerase amino acid sequence as shown in SEQ ID NO.1, and comprises at least one of amino acid mutation L345E, L352D and S438M. The Taq DNA polymerase mutant has excellent tolerance to urea, and solves the dual technical problems that complex biological samples containing endogenous inhibitors such as urea can be efficiently amplified, and the Taq DNA polymerase mutant is compatible with difficult PCR reactions which take urea as an additive to optimize a high GC template and the like. Therefore, the mutant is a molecular biology tool enzyme which is wider in applicability and more stable and reliable in performance.

The invention discloses a quinoa CqARF2 gene mutant as well as an identification method and application thereof. Chenopodium quinoa seeds are mutated on a large scale by utilizing a chemical mutagenic agent EMS, a CqARF2 gene function deletion mutant arf2 is adopted as a quinoa mutant, G at the 1050th site of the CqARF2 gene of the mutant is mutated into A, and amino acid at the 350th site is mutated into a termination codon, so that the translation process of CqARF2 protein is terminated in advance. The chenopodium quinoa CqARF2 gene function deletion mutant arf2 shows remarkable agronomic character improvement, seeds are large and full, compared with a wild type, the hundred-grain weight of the mutant seeds is increased by about 40%, the seed diameter is increased by about 10%, the seed thickness is increased by about 30%, and a certain foundation is laid for breeding new high-yield and high-quality chenopodium quinoa varieties.

The invention relates to the field of tumor cell therapy, in particular to application of T lymphocyte chimeric with a B7-H3 receptor to treatment of head and neck tumors, and provides an anti-B7-H3 scFv, the scFv comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an HCDR region and an HFR region, and the light chain variable region comprises an LCDR region and an LFR region; compared with a wild type scFv sequence, the scFv sequence has the advantage that a plurality of amino acids with positive charges in the HFR region and / or the LFR region are mutated into amino acids without charges. According to the invention, positive charge plaques on the CAR surface of an scFv sequence of a B7-H3 human-derived monoclonal antibody MGA271 are changed in a charged amino acid mutation manner, so that a B7-H3. CAR-T cell is optimized, and it is proved that the B7-H3. CAR-T cell optimized by PCP can effectively kill B7-H3 positive tumor cells in vivo and in vitro; and a new method and thought are provided for clinical targeted treatment of B7-H3 positive solid tumors.

The present invention provides an α-amino acid ester acyltransferase mutant and its application. The α-amino acid ester acyltransferase mutant includes: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; (b) a protein having an amino acid mutation at at least one of the following sites in the amino acid sequence in (a): P158, K80, T208, A302, A175, N85, N605, K346, S225, I34, or S348, and having the function of an α-amino acid ester acyltransferase; or (c) a protein having more than 80% homology with the amino acid sequence defined in (a) or (b) and having the function of an α-amino acid ester acyltransferase. The α-amino acid ester acyltransferase mutant of the present invention has the advantages of a broad substrate spectrum and high catalytic activity, can be well used in the synthesis of oligopeptides, has low cost and high yield, and realizes true green chemistry.

The invention belongs to the technical field of bioengineering, and particularly relates to a zearalenone toxin degrading enzyme variant and application thereof. The amino acid sequence of the zearalenone toxin degrading enzyme is as shown in SEQ ID NO.2, or the zearalenone toxin degrading enzyme with the amino acid sequence as shown in SEQ ID NO.2 is obtained through amino acid mutation. The zearalenone toxin degrading enzyme mutant is obtained through mutation screening, and compared with a wild type, the zearalenone toxin degrading enzyme mutant has the advantages that the zearalenone degrading capability is obviously improved; besides, the zearalenone toxin degrading enzyme is expressed by using alfalfa, the zearalenone toxin degrading enzyme with biological activity is easy to obtain, and the zearalenone toxin degrading enzyme has certain application potential in prevention and treatment of animal poisoning caused by zearalenone toxin pollution in agriculture and animal husbandry production.

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.

Disclosed herein are predictive models for T-cell epitope prediction, B-cell epitope prediction, and protein design wherein a method is implemented for generating a protein variant amino acid sequence of a target protein having one or more modified properties, the method comprising: (a) iteratively sampling an input amino acid sequence of the target protein, and (b) sampling the individual protein score of at least one weighted relative contribution of the single residue mutant input amino acid sequence to the at least one target property across a plurality of other single residue mutant input amino acid sequences to generate a combined protein score, wherein the combined protein score corresponds to the protein variant comprising one or more amino acid mutations of the single residue mutant input amino acid sequences.

The invention discloses a synthesis method of an insulin analogue, which comprises the following steps of: firstly, obtaining A-chain peptide resin by using a solid-phase synthesis method, constructing a disulfide bond between A6 and A11 on a solid phase, and cracking the A-chain peptide resin by using a cracking solution containing a sulfydryl activator to obtain a peptide chain with an activated sulfydryl group and a protected sulfydryl group; then a solid-phase synthesis method is used for obtaining B-chain peptide resin, Lys side chain protecting groups are removed on a solid phase in a fixed-point mode, side chain fatty acid modification is completed, and a peptide chain with an exposed sulfydryl group and a protected sulfydryl group is obtained after cracking is conducted through a cracking solution; and mixing the products in a solution according to a certain proportion to form a first pair of intermolecular disulfide bonds, and constructing and purifying the last pair of disulfide bonds to obtain the insulin analogue. The method disclosed by the invention has the advantages of convenience in synthesis, low cost, simple process and the like, can be used for research on amino acid mutation of insulin compounds, and has important practical significance on development of insulin drugs.

The present invention relates to the technical field of biomedicine. Specifically provided are a respiratory syncytial virus (RSV) Pre-F recombinant protein nanoparticle vaccine and a preparation method therefor. By performing amino acid mutation on an RSV Pre-F protein, and performing fusion expression on the mutated Pre-F protein and ferritin particles in eukaryotic cells, PreF-Ferritin fusion protein nanoparticles, in which eight Pre-F protein trimers are densely displayed on the surface of each ferritin particle, are obtained. By stabilizing and exposing antigen epitopes required to be displayed and disrupting or masking unrequired antigen epitopes, the PreF-Ferritin fusion protein nanoparticles effectively improve the immunogenicity and the production stability of the antigen. Experiments show that: when the PreF-Ferritin fusion protein is injected into mice, high-titer protective sera can be acquired, and the mice sera can yield relatively high neutralizing titers against RSV.

The invention relates to the technical field of nucleic acid detection, and discloses a detection method based on LAMP (loop-mediated isothermal amplification) and CRISPR (clustered regularly interspaced short palindromic repeats) / Cas and application. The detection method comprises the following steps: carrying out key amino acid mutation on a PAM structural domain of an eBrCas12b protein; the preparation method comprises the following steps: preparing Cas12b sgRNA through in-vitro transcription and purification; the cis-cleavage capability of the eBrCas12b, the eBrCas12b-G and the eBrCas12b-GN on the target sequence is evaluated, and the cis-cleavage capability of the Escherichia coli carrying pUC19-blaNDM5 plasmids is constructed, and an eBrCas12b-GN one-tube method detection system is applied to detection of the NDM drug-resistant escherichia coli. According to the detection method based on LAMP and CRISPR / Cas and the application, a traditional LAMP-Cas12b one-tube method detection system is optimized, and the sensitivity and speed of one-tube method detection are remarkably improved.

The invention provides a BFD mutant and application thereof as a carbon chain elongase, and the BFD mutant is characterized in that (a) compared with an amino acid sequence as shown in SEQ ID NO.1, the BFD mutant contains at least one of the following sites: L43, A73, T100 and K165, and an amino acid sequence which is subjected to amino acid mutation and has an active function of the carbon chain elongase; or (b) an amino acid sequence which is obtained by substitution and / or deletion and / or addition of one or more conserved amino acid residues of the BFD mutant and the amino acid sequence shown in (a) and has the same function. The enzyme activity of the BFD mutant is obviously improved, and the yield and conversion rate of glycolaldehyde and / or 1, 3-dihydroxyacetone generated from formaldehyde are improved.

The invention belongs to the technical field of gene engineering and enzyme engineering, and discloses a glutaminase mutant, immobilized glutaminase and application thereof. The glutaminase mutant is based on an amino acid sequence as shown in SEQ ID NO.1 and is subjected to amino acid mutation at the following sites: E113K, R136M and H223V, and the amino acid sequence of the glutaminase mutant is as shown in SEQ ID NO.3. The immobilized glutaminase is prepared by using the glutaminase mutant, the enzyme activity is increased by 5-10 times compared with that of a wild type, the substrate concentration in the L-theanine catalyzed and synthesized by the immobilized glutaminase reaches 80-150g / L, the conversion rate is greater than 99%, the L-theanine can be recycled through simple filtration, and the L-theanine can be repeatedly used for more than five times after being recycled. And the conversion rate during repeated use is basically the same as that during primary use, so that the method has relatively high industrial application potential.

According to the protein thermal stability point mutation prediction model construction method disclosed by the invention, multi-dimensional physical quantity selection is performed within the preset distance range around the target residue, so that the structural characteristics of the protein can be reflected more comprehensively, and a richer information basis is provided for predicting the protein thermal stability point mutation. When the three-dimensional convolutional neural network is trained, interaction between atoms in a large range can be captured through a large-size convolution kernel, and a complex relation is revealed; local details can be focused through a small-size convolution kernel, structural features can be comprehensively described from different scales through combination of the two, and it is ensured that the model learns key information. The normalization method of the transfer learning neural network emphasizes information provided by experimental data, keeps the benchmark probability of non-mutated amino acid as a reference, can more accurately predict the influence of amino acid mutation on the thermal stability and functional activity of the protein, and especially, can predict the thermal stability and functional activity of the protein under the condition of lacking comprehensive experimental data. Reasonable inference can still be made depending on known experimental information and reference values.

A varicella-zoster virus (VZV) nanoparticle protein, and a preparation method therefor and a use thereof. The VZV protein comprises a partial or full sequence of an amino acid sequence of an extracellular region of a VZV gE glycoprotein, with W at position 200 mutation to C and L at position 245 mutation to C in the amino acid sequence of the extracellular region of the VZV gE glycoprotein. By means of the rational optimization design of the amino acid sequence of the VZV gE protein by means of protein genetic engineering, the VZV gE recombinant protein modified with amino acid mutations has increased stability and immunogenicity compared with the VZV gE protein. Moreover, by further designing the protein structure, the VZV gE protein is repeatedly displayed on the surface of ferritin nanoparticles with the desired epitopes exposed, thereby further enhancing immunogenicity.

The invention discloses an anti-inhibition Taq DNA polymerase and application thereof, and relates to the technical field of biology. According to the anti-inhibition Taq DNA polymerase, amino acid mutations of K206R, F667Y and the like are carried out on an amino acid sequence of a wild type Taq DNA polymerase with an amino acid sequence as shown in SEQ ID No.1, so that a certain degree of anti-inhibition property is obtained. In addition, the invention also discloses application of the anti-inhibition Taq DNA polymerase in preparation of reagents or kits, and the like. Compared with a wild type Taq DNA polymerase, the anti-inhibition Taq DNA polymerase can maintain the PCR amplification capability in the presence of an inhibitor such as polysaccharide.

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 provides a glutaminase mutant and application of the glutaminase mutant. Wherein the glutaminase mutant comprises (a) a protein which is subjected to amino acid mutation and has a glutaminase activity function on at least one of the following sites of S467, Y334, K400, N521, R337, E402 or D447 of an amino acid sequence as shown in SEQ ID NO: 2, and (b) a protein which is subjected to amino acid mutation on at least one of the following sites of S334, Y334, K400, N521, R337, E402 or D447 of the amino acid sequence as shown in SEQ ID NO: 2; and (b) a protein which has 80% or more homology with the amino acid sequence limited by (a) and has a glutaminase function. The glutaminase mutant with the amino acid sequence disclosed by the invention has relatively high specific enzyme activity during enzyme catalytic reaction, and is low in cost and strong in market competitiveness when being used for commercial food processing.

A cage-like nanocarrier for targeted delivery of siRNA, and a preparation method therefor and the use thereof. The preparation method comprises: (A) mutating a negatively charged or uncharged amino acid on the inner surface of a ferritin into a positively charged amino acid; and any one or more of the following steps: (B) coupling the N-terminus of the ferritin to a functional peptide having nucleic acid affinity; (C) coupling the N-terminus of the ferritin to a functional peptide promoting lysosomal escape; (D) truncating the E-helix at the C-terminus of the ferritin; (E) coupling the C-terminus of the ferritin to a functional peptide having nucleic acid affinity; and (F) coupling the C-terminus of the ferritin to a functional peptide promoting lysosomal escape. A new nucleic-acid-loaded protein nanocage carrier is constructed by means of modifying a negatively charged inner cavity of ferritin to make same positively charged. By means of electrostatic adsorption, a negatively charged siRNA can be efficiently loaded into a ferritin nanocage, thereby significantly improving the in-vivo and in-vitro delivery stability of siRNA, lysosomal escape functions, and efficacy of targeted therapy.

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 provides an aspartic protease heat-resistant mutant and application thereof. Wherein the aspartic protease heat-resistant mutant comprises (a) a protein which is based on wild type aspartic protease pap1 as shown in SEQ ID NO: 1, is subjected to amino acid mutation on at least one of the following sites: G133, D159, S376, T198, T103, T144, L303 or A387, has aspartic protease activity and has higher heat resistance than that of the wild type aspartic protease pap1; or (b) a protein which has more than 80% of homology with the amino acid sequence limited in (a), has aspartic protease activity and has heat resistance higher than that of wild type aspartic protease pap1. The aspartic protease can solve the problem of poor heat resistance of aspartic protease in the prior art, and is suitable for the field of enzyme catalysis.

This invention belongs to the field of enzyme engineering technology and discloses a BhrPETase mutant and its applications. The invention provides a BhrPETase mutant with the amino acid sequence shown in SEQ ID No. 2, designated as mutant F208I. Compared to the wild-type mutant BhrPETase, this mutant exhibits a 1.39-fold increase in PET degradation activity. Based on mutant F208I, this invention further rationally designs amino acid mutations, providing 21 BhrPETase mutants. The BhrPETase mutants provided by this invention, while maintaining thermal stability essentially equivalent to wild-type BhrPETase, increase PET degradation activity by 1.39 to 4.24 times. The BhrPETase mutants provided by this invention can be applied in fields such as PET degradation, PET degradation product recovery, or the preparation of PET degradation agents.

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.

The invention provides an L protein mutated recombinant foot-and-mouth disease virus strain and application thereof in vaccine preparation, and belongs to the technical field of biological medicine. Firstly, it is found that overexpression of CISD2 protein in host cells can inhibit FMDV replication, and an inhibition mechanism is analyzed, specifically, the CISD2 inhibits FMDV replication by inhibiting expression of L protein, especially inducing degradation of the L protein through a proteasome pathway. The invention also identifies a key site for degrading the L protein by the CISD2 protein, the recombinant foot-and-mouth disease virus with the mutated key amino acid site of the L protein is successfully constructed and rescued by introducing amino acid mutation of the key site into the L protein by utilizing a reverse genetic technology, the capabilities of degrading the L protein and inhibiting FMDV replication by the CISD2 protein are eliminated, the virus titer is improved, and the recombinant foot-and-mouth disease virus can be used for preparing the recombinant foot-and-mouth disease virus with the mutated key amino acid site of the L protein. The strain has good production performance and can be used for preparing recombinant foot-and-mouth disease virus strains and recombinant vaccine strains.

The invention provides a method for preparing (R)-4-chloro-3-hydroxybutyric acid ethyl ester ((R)-CHBE) through enzyme catalysis, which comprises the following steps: isopropanol is used as a hydrogen donor, a ketoreductase mutant is used for carrying out catalytic reduction reaction of COBE to prepare (R)-CHBE, and the ketoreductase mutant has amino acid mutation at the 83rd site on the basis of an amino acid sequence shown as SEQ ID NO.1. The invention also provides a preparation method of (R)-4-chloro-3-hydroxybutyric acid ethyl ester ((R)-CHBE). The thermal stability, namely heat resistance, of the ketoreductase mutant is improved, and by combining with the use of a rectifying tower, the method disclosed by the invention improves the utilization rate of isopropanol and can discharge a byproduct acetone in time, so that the substrate conversion rate and the product concentration are improved. The invention also provides an enzyme catalysis device used in the method.

The invention relates to the technical field of biological detection, and particularly discloses an ELISA kit for detecting a neutralizing antibody of a severe fever with thrombocytopenia syndrome virus and an application method of the ELISA kit. The ELISA kit comprises an ELISA plate on which SFTSV receptor LRP1 protein is fixed, and an enzyme-labeled SFTSV Gn-8M, wherein the SFTSV receptor LRP1 protein is fixed on the ELISA plate; the SFTSV Gn-8M is an SFTSV membrane protein Gn mutant designed by the invention, and can be specifically combined with an SFTSV receptor LRP1 protein on an elisa plate, and compared with a standard virus strain Gn protein, the self-designed SFTSV Gn-8M has amino acid mutation of eight sites, has stronger combining capacity with a host cell receptor, and can be used for detecting the SFTSV. The kit disclosed by the invention has the characteristics of high safety, high efficiency, high flux, high specificity, high sensitivity, high stability and the like, and is suitable for large-scale rapid screening of the neutralizing antibody of the severe fever with thrombocytopenia syndrome virus.