462 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 provides a sweet protein mutant as well as a preparation method and application thereof. The mutant is obtained by amino acid mutation on the basis of an amino acid sequence as shown in SEQ ID NO.1 of sweet protein. The amino acid mutation type comprises at least one of K5D, V7R, D29N or E53R. The mutation is introduced into the amino acid sequence of the sweet protein, so that compared with the sweet protein corresponding to the original sequence, the sweetness of the mutant is obviously improved.

The invention discloses a milk-derived broad-spectrum antibacterial peptide and application thereof, and relates to the technical field of biology. The amino acid sequence of the antibacterial peptide is as shown in any one of SEQ ID NO.2-6. Five novel antibacterial peptides are obtained by carrying out symmetrical structure design and amino acid mutation on the basis of a template peptide, and tests find that the five novel antibacterial peptides have broad-spectrum antibacterial activity which is obviously higher than that of the template peptide, and basically have no cytotoxicity. Wherein the geometric mean (GM) of the minimum inhibitory concentration can reach 3.60, and the therapeutic index (TI) can reach 71.11. The antibacterial peptide prepared by the invention still keeps good antibacterial activity in physiological salt concentration and protease environment, has certain serum stability, and has higher application value; in addition, the antibacterial peptide has a good inhibition effect on the formation of a bacterial biofilm, and has a synergistic effect with traditional antibiotics.

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 relates to a T7 RNA polymerase mutant and application thereof. Specifically, the invention discloses a T7 RNA polymerase mutant, and the polymerase mutant has at least one amino acid mutation at sites corresponding to a wild type T7 RNA polymerase and selected from the following groups: the 44th site, the 45th site, the 46th site, the 172nd site, the 389th site, the 425th site, the 441st site, the 632th site, the 643th site, the 754th site and the 789th site; the serial number of the mutation site is based on the sequence shown in SEQ ID NO: 1, the mutation leads to obvious reduction of a byproduct dsRNA generated in the process of enzyme synthesis of mRNA, and meanwhile, the generation of RNA aggregates is reduced.

The invention relates to the field of biochemical engineering, in particular to an alcohol dehydrogenase mutant and application thereof. The mutant is obtained by mutating alcohol dehydrogenase with an amino acid sequence encoding SEQ ID NO: 4 at one or more sites selected from 62nd, 108th, 130th, 132, 134, 182nd, 206th, 287th, 303th, 313th and 314, and the mutation is that the amino acid at the site is mutated into one of A, G, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R and H. According to the alcohol dehydrogenase mutant and the application of the alcohol dehydrogenase mutant, the feeding amount of substrates is increased; the high conversion rate (gt; 99%) of the substrate is realized; according to the present invention, the new YqjM has the good product ee value (gt; 98%), and it is determined that the YqjM derived from the bacillus subtilis old yellow enzyme (belonging to ketene reductase) has the optimal catalytic activity, such that the good industrial application performance is provided.

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 invention relates to a mutant of an RSV pre-fusion conformation F protein, the mutant comprises amino acid mutation relative to an amino acid sequence of a wild type RSV F protein, the amino acid mutation is that amino acids at positions 392 and 493 or near the 493 are substituted by C, and a disulfide bond is formed between mutated positions 392C and 493C or near the 493C. The invention also provides a related DNA (deoxyribonucleic acid) coding sequence, a recombinant plasmid, an mRNA (messenger ribonucleic acid) sequence and a corresponding RSV vaccine. By introducing disulfide bonds at proper positions and combining with cavity filling, electrostatic mutation, proline mutation and the like, the expression and stability of the F protein with conformations before and after fusion and trimerization conformations are remarkably improved, and the F protein is remarkably inhibited from being converted into conformations after fusion. The mutant is also applied to preparation of RSV vaccines and detection of respiratory syncytial virus infection.

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.

A nitrile hydratase mutant and the use thereof. The nitrile hydratase mutant is a wild-type nitrile hydratase having a mutation of an amino acid residue in the flexible loop domain, and comprises any one of or a combination of mutations 1-3, wherein mutation 1 is a polar amino acid mutation located in the flexible loop between secondary domains S1 and S2 of the β subunit of the wild-type nitrile hydratase; mutation 2 is a polar amino acid mutation located in the flexible loop between secondary domains H6 and H7 at the starting end of the β subunit of the wild-type nitrile hydratase; and mutation 3 is a hydrophobic amino acid mutation in starting flexible loop L1 of the β subunit of the wild-type nitrile hydratase. On the basis of the wild-type nitrile hydratase, the nitrile hydratase mutant can comprise any one of or a combination of the mutations described above, which can exert regulatory effects alone or synergistically, has a certain enzyme activity, and has good thermal stability and tolerance.

The invention discloses a transformation method and application of a G protein coupled receptor, and belongs to the technical field of protein engineering. In order to improve the molecular weight of membrane protein, enable the enlarged part of the membrane protein and the original membrane protein to form a relatively rigid structure and further meet the structural analysis requirement of a cryoelectron microscope, the invention provides a method for transforming a G protein coupled receptor. The method comprises the following steps: co-expressing fusion protein GPCR (Glycine Polymerase Chain Reaction)-GFP10-11 and GFP1-9 fragments formed by GPCR and GFP10-11 fragments in a eukaryotic cell expression system, and adding GFP-clamp in the purification process; one amino acid at the specific site of GPCR is mutated into cysteine, glutamic acid at the 186th position of the amino acid sequence of GFP-clamp is mutated into cysteine, and a disulfide bond is formed between the amino acid and glutamic acid. The molecular weight of the modified G protein coupled receptor obtained by using the method can be increased to 85 kD or above, and the rigidity of the whole compound space structure is relatively strong, so that the modified G protein coupled receptor can be used for structural analysis of a cryoelectron microscope.

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 present invention relates to an adeno-associated virus (AAV) capsid protein variant having an amino acid mutation at the 328th to 331st amino acid residues of the AAV capsid protein relative to the 328th to 331st amino acid residues of the parent AAV capsid protein, for example, an amino acid sequence as shown in SEQ ID NO: 4 to replace the 328th to 331st amino acid residues of the parent AAV capsid protein wherein the amino acid position is numbered according to SEQ ID NO: 5. The invention also relates to an isolated nucleic acid encoding the AAV capsid protein variant, a nucleic acid vector comprising the same, and a host cell. The present invention also relates to a recombinant adeno-associated virus (rAAV) vector comprising said capsid protein variant, a pharmaceutical composition comprising said rAAV vector, and uses thereof in the treatment or prevention of skeletal muscle diseases.

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 belongs to the technical field of enzyme engineering, and discloses a high-activity and high-stability PET hydrolase mutant and application thereof. The high-activity and high-stability PET hydrolase mutant is a protein A, a protein B or a protein C, wherein the protein A is obtained by mutating the 132 amino acid of wild cutinase TfCut from glutamine Q to lysine K; the protein B is obtained by mutating the 244th and 293rd amino acids into cysteine C on the basis of the protein A; protein C is a fusion protein containing protein A or protein B. The modified enzyme breaks through a trade-off mechanism of enzyme catalytic activity and stability, and the activity and thermal stability of the modified enzyme are greatly improved, so that the industrial application value of the PET hydrolase in the aspect of PET degradation is improved.

Provided is a mutant of a respiratory syncytial virus (RSV) pre-fusion conformational F protein. The mutant comprises an amino acid mutation relative to the amino acid sequence of a wild-type RSV F protein, the amino acid mutation being amino acids at a position 392 and a position 493 or a position near the position 493 being substituted by C, and a disulfide bond being formed between a position 392C and a position 493C or a position near the position 493C after the mutation. Also provided are a related DNA coding sequence, recombinant plasmid and mRNA sequence and a corresponding RSV vaccine. By introducing a disulfide bond at a suitable position, and combining same with cavity filling, electrostatic mutation, proline mutation and the like, the expression and stability of an F protein having pre- and post-fusion conformations and a trimerized conformation are significantly improved, and the conformation of the F protein is significantly inhibited from being converted into the post-fusion conformation. The mutant is also applied to preparation of an RSV vaccine and RSV infection detection.

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.

Provided herein is a ribonucleic acid (RNA) encoding a spike (S) protein or an immunogenic fragment thereof of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising at least one non-naturally occurring amino acid mutation. In some embodiments, the S protein is derived from a delta variant. Additionally provided are relevant polynucleotides, vectors, cells, compositions, kits, production methods and methods of use.

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 belongs to the technical field of biology, and relates to zymoprotein, in particular to recombinant arthrobacter globiforme uricase and application thereof. The recombinant arthrobacter globiforme uricase has single-site amino acid mutation, amino acids are located in an active region and an inactive region of the uricase, and the amino acid sequence of the recombinant arthrobacter globiforme uricase is shown as any one of SEQ ID NO: 1-8. Compared with wild uricase, the mutant uricase formed after amino acid is introduced into uricase at a fixed point has higher activity, and the use amount of uricase can be more effectively reduced under the condition that the content of uric acid is effectively controlled.

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 relates to a dammarendiol synthase mutant and application of the dammarendiol synthase mutant in preparation of beta-amyrin alcohol, and belongs to the technical field of genes. The dammarendiol synthase mutant is subjected to the following amino acid mutation based on a sequence as shown in SEQ ID NO.1: T128L, and other amino acid sequences are kept unchanged; or the dammarendiol synthase mutant is subjected to the following amino acid mutation on the basis of the sequence as shown in SEQ ID NO: 1: T128L, L242M, F253L, F255M, V327L, A377V, V485Q and A493G, and other amino acid sequences are kept unchanged. According to the invention, reverse mutation modification is carried out through the difference sites, so that DDS has a catalytic function of beta-AS, and further deep analysis is carried out on the catalytic difference formed by DDS and beta-AS in the evolution process.

The invention discloses a stable pre-fusion RSV F protein and application thereof, the RSV F protein comprises one or more amino acid mutations to stabilize the RSV F protein in a pre-fusion conformation, and the RSV F protein has high affinity and neutralizing activity. Also disclosed are fusion proteins or multimers, immunogens, virus-like particles or protein nanoparticles comprising the RSV F protein, as well as nucleic acid molecules encoding the same, vectors, host cells, and immunogenic compositions.