26 results about "Ligase activity" patented technology

The present application provides: a microorganism with enhanced lipoate protein ligase activity; a composition for producing histidine comprising the microorganism; and a method for producing histidine, the method comprising a step for culturing the microorganism.

The invention particularly relates to a determination method of DNA ligase and a related compound. The determination method comprises the following steps that a single-chain sequence MB is provided, at least the 5'end and the 3 'end of the single-chain sequence MB are DNA sequences and are complementarily paired, a neck ring structure can be formed, and the 5' end and the 3 'end of the single-chain sequence MB are provided with a fluorophore and a quenching group respectively; providing two single-stranded DNA sequences D1 and D2, wherein the two single-stranded DNA sequences D1 and D2 are complementary and adjacent to the annular part of the single-stranded sequence MB; and hybridizing the two single-stranded DNA sequences D1 and D2 with the single-stranded sequence MB to form a compound with a neck ring. Respectively treating the compound by using a DNA ligase standard substance to obtain a standard curve, and substituting the detection result of the DNA ligase to be detected into the standard curve. The method is simple to operate and does not have a fluorescence background value, the activity of the ligase can be judged by directly depending on the intensity of the generated fluorescence, and the comparison is more convenient and more sensitive.

The present disclosure provides a microorganism with increased activity of lipoate protein ligase, a composition for producing pantothenic acid and / or pantoic acid comprising the microorganism, and a method for producing pantothenic acid and / or pantoic acid comprising a step of culturing the microorganism, wherein the microorganism has excellent pantothenic acid and / or pantoic acid producing ability.

The present invention provides a polypeptide ligase mutant and a method for preparing a polypeptide. The polypeptide ligase mutant comprises: (a) a protein mutated from the amino acid sequence shown in SEQ ID NO: 1, the mutation comprising a mutation occurring at the S307 site; or (b) a protein having 70% or more identity with the amino acid sequence defined in (a) and having polypeptide ligase activity. The present invention can solve the problem in the prior art of poor activity of polypeptide ligases, and is applicable to the field of polypeptide synthesis.

A method for producing γ-Glu-Val-Gly is described, wherein the method includes the steps of cultivating a γ-Glu-Val-Gly-producing bacterium belonging to the family Enterobacteriaceae in a culture medium so that the γ-Glu-Val-Gly accumulates in the culture medium or the cells of the bacterium, or both, and collecting the γ-Glu-Val-Gly from the culture medium or the cells of the bacterium, or both. The bacterium has been modified to overexpress a gene encoding a protein having L-threonine 3-dehydrogenase activity and a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity.

Provided herein are compositions, kits, systems, and methods employing single-stranded end-preserving adaptors. Such single-stranded adaptors are attached to DNA duplex molecules while preserving original 5′ or 3′ single-strand protruding ends (e.g., present in cell-free DNA) by attaching such adapters to 3′ ends the DNA duplex molecules using a single strand ligase that has step 3 ligase activity, but not step 2 adenylyl transfer activity, and attaching such adapters to 5′ ends of the DNA duplex molecules using a ligase enzyme (e.g., a circligase), thereby forming loop-like structures on one or each end of the DNA duplex molecules. In further embodiments, the loop-like structures are cleaved (e.g., by an endonuclease) as the single-stranded adapters have a cleavable portion, thereby generating a two-part adapter on one or both ends of the DNA duplex molecules that preserves the initial 5′ or 3′ single-strand protruding ends, along with any methylation present.

To provide methods for degrading target surface proteins in the ubiquitin pathway using bispecific binding agents or immunoconjugates that bind to the target surface proteins and membrane-bound ubiquitin E3 ligase.SOLUTION: The present invention relates, inter alia, to methods of degrading target surface proteins in the ubiquitin pathway using bispecific binding agents or immunoconjugates that bind to target surface proteins and membrane-bound ubiquitin E3 ligase. The present invention also relates to methods of degrading target surface proteins in the ubiquitin pathway using engineered membrane spanning proteins that bind to target surface proteins and exhibit ubiquitin E3 ligase activity. The present disclosure also provides compositions and methods useful for making bispecific binding agents and engineered transmembrane proteins, immunoconjugates, nucleic acids encoding them, host cells genetically modified with said nucleic acids, and methods of modulating the activity of cells and / or treating various diseases, such as cancer.SELECTED DRAWING: Figure 1

The application provides a polypeptide ligase mutant and a polypeptide preparation method. The polypeptide ligase mutant comprises: (a) a protein which is mutated based on the amino acid sequence shown in SEQ ID NO: 1, and the mutation comprises a mutation at a S307 site; or (b) a protein which has more than 70% homology with the amino acid sequence defined in (a) and has polypeptide ligase activity. The polypeptide ligase activity in the prior art can be improved, and the polypeptide ligase mutant is suitable for the field of polypeptide synthesis.

Provided herein are compositions, kits, systems, and methods employing single-stranded end-preserving adaptors. Such single-stranded adaptors are attached to DNA duplex molecules while preserving original 5' or 3' single-strand protruding ends (e.g., present in cell-free DNA) by attaching such adapters to 3' ends the DNA duplex molecules using a single strand ligase that has step 3 ligase activity, but not step 2 adenylyl transfer activity, and attaching such adapters to the 5' ends of the DNA duplex molecules using a ligase enzyme (e.g., a circligase), thereby forming loop-like structures on one or each end of the DNA duplex molecules. In further embodiments, the loop-like structures are cleaved (e.g., by an endonuclease) as the single-stranded adapters have a cleavable portion, thereby generating a two-part adapter on one or both ends of the DNA duplex molecules that preserves the initial 5' or 3' single-strand protruding ends, along with any methylation present.

The invention belongs to the technical field of biology, in particular to the technical field of biomedicine, and particularly relates to a method for improving fidelity of DNA and RNA and DNA-RNA mixed connection based on selenium modified ATP and application, ATP alpha Se is used for enhancing the mismatch recognition capacity of DNA ligase and RNA ligase, and ATP alpha Se can improve the connection accuracy of multiple ligase. According to the present invention, ATP [alpha] Se is used, the key selective interaction of the ligase active site is specifically improved through the selenium atom, the ligase is sensitive to the substrate mismatch of the large expansion region, and the fidelity of the four-base protruding terminal connection in almost all the connection sequences is improved by using the ATP [alpha] Se technology; the ATP alpha Se is used, so that the accuracy of Golden Gate Assay is improved, the accuracy of simultaneous assembly of multiple fragments is improved, the success rate is improved, and the cost of genome assembly can be reduced; compared with the prior art, the kit has the advantages that the kit has the advantages that the kit can be used for detecting multiple SNP genes, false positive in multiple ligation-dependent probe amplification (MLPA) reaction can be effectively inhibited, the accuracy of gene detection with multiple SNPs at the same time can be higher, and the cost of simultaneous detection of multiple genes can be reduced.

To provide isolated polypeptides having Asx-specific ligase and cyclase activity.SOLUTION: Disclosed is an isolated polypeptide having protein ligase activity, preferably cyclase activity, the isolated polypeptide comprising a specific sequence. Enzymes having Asx-specific ligase and cyclase are isolated from plants of the Violaceae family.SELECTED DRAWING: None

In one aspect, the invention relates to substituted 1-phenyl-3-(piperidin-4-yl)urea analogs, derivatives thereof, and related compounds, which are useful as inhibitors of the DCN1-UBC12 interaction inhibitors of DCN1-mediated cullin-RING ligase activity, methods of making same, pharmaceutical compositions comprising same, methods of treating disorders using the disclosed compounds and compositions, methods of treating disorders associated with a DCN1-UBC12 interaction dysfunction, methods of treating disorders associated with a DCN1-mediated cullin-RING ligase activity dysfunction, methods of male contraception comprising the disclosed compounds and compositions, and kits comprising the disclosed compounds and compositions. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

Various embodiments relate generally to the field of enzyme technology and specifically relate to polypeptides having Asx-specific protein ligase and cyclase activity and to nucleic acids encoding those as well as methods of the manufacture of said enzymes, more particularly methods of producing stable and constitutively active protein ligases.

This invention discloses a short peptide and its derivatives that mimic the N-terminus of RhoE, and their pharmaceutical applications in the treatment of myocardial hypertrophy, belonging to the field of biomedical technology. This short peptide precisely mimics the amino acid sequence from position 1 to 20 of RhoE, specifically binding to the WW domain of WWP2 and competitively blocking the interaction between the N-Loop and C-Loop of its HECT domain. This causes the WWP2 conformation to change from a self-inhibited "closed" state to an activated "open" state, activating E3 ligase activity. The short peptide retains its binding ability to the cardiomyocyte membrane cavernin CAV3, allowing it to efficiently enter the cardiomyocyte cytoplasm via the cavernin endocytosis pathway, overcoming the deficiency of traditional drugs in clearing intracellular pathogenic proteins. Peptide derivatives constructed by fusing transmembrane peptides such as TAT ​​and T7 further improve intracellular delivery efficiency. Experiments have demonstrated that this short peptide and its derivatives can significantly promote WWP2 self-ubiquitination, enhance cardiomyocyte autophagic flux, clear intracellular pathogenic proteins, and effectively inhibit cardiomyocyte hypertrophy, making it suitable for the preparation of anti-myocardial hypertrophy drugs.

The invention discloses a ubiquitin ligase TRIM15 inhibitor and application thereof, and belongs to the technical field of medicinal chemistry. Researches show that TRIM15 plays a role in various cell processes, especially in ubiquitination, immune regulation and cell stress reactions, the invention screens and identifies the compound shown in the following formula, or the use of the hydrate, solvate, pharmaceutically acceptable salt, pharmaceutically acceptable ester or prodrug thereof in preparation of drugs, the medicine is used as an ubiquitin ligase TRIM15 inhibitor for inhibiting the activity of the ubiquitin ligase TRIM15; researches confirm that the ubiquitin ligase TRIM15 inhibitor shows an obvious anti-tumor effect in vivo and in vitro, is expected to be applied to the fields of anti-tumor, immunoregulation and the like, and is combined with the existing clinical medicines to improve the treatment effect.

The invention belongs to the technical field of medicines, and particularly relates to application of indole-3-carbinol in preparation of thyroid cancer immunotherapy drugs. Through cell and animal experiments, the indole-3-methanol can be used for remarkably enhancing the curative effects of thyroid cancer immunotherapy and targeted therapy by inhibiting the activity of WWP1 ubiquitin ligase, stabilizing STAT6 protein expression, inhibiting TDO2-mediated tryptophan metabolism and remodeling an immune microenvironment. Indole-3-A is clear in action mechanism and high in safety, and has important clinical application value. In a mouse model, single use of indole-3-carbinol can significantly inhibit thyroid cancer growth and enhance CD8 + T cell infiltration and granzyme B activity, and the effect of indole-3-carbinol is equivalent to that of a PD-1 monoclonal antibody; the combination of indole-3-methanol and PD-1mAb can synergistically enhance the anti-tumor immune response and significantly inhibit tumor growth. The pharmaceutical composition is especially suitable for immunotherapy of drug-resistant or advanced refractory cases, and shows a remarkable synergistic effect compared with the existing therapy.

Disclosed herein are compounds, compositions, and methods for inhibiting HECT E3 ubiquitin ligases. In some embodiments, the compounds are formulated as a pharmaceutical composition and administered to a subject in need thereof. In some embodiments, the subject in need thereof is diagnosed with a neurological disease or a cancer characterized by increased or ectopic HECT E3 ligase activity.

The present application provides a polypeptide ligase mutant and a polypeptide preparation method. The polypeptide ligase mutant includes (a) a protein with a mutation based on the amino acid sequence shown in SEQ ID NO: 1, and the mutation includes a mutation at the S297 site; or (b) a protein with more than 70% homology with the amino acid sequence defined in (a) and having polypeptide ligase activity. The polypeptide ligase mutant can solve the problem of poor connection activity of the polypeptide ligase in the prior art and is suitable for the field of polypeptide synthesis.

This invention discloses a T4 RNA ligase 2 mutant, its preparation method, and its applications, relating to the fields of biology and enzyme engineering. The T4 ligase 2 mutant provided by this invention has mutations at at least one of the following positions in the wild-type T4Rnl2 sequence: positions 62, 103, 166, 168, 193, 217, 260, 297, 298, 303, 306, 311, 313, and 318. Compared to the wild type, the mutant exhibits significantly improved thermostability and maintains high ligase activity at higher temperatures, such as 45°C and 50°C. In addition to ligating native nucleic acid sequences, the mutant can also ligate modified non-native nucleic acids at high temperatures. The mutant provided by this invention enables high-temperature synthesis of ribonucleic acid, effectively reducing the generation of byproducts due to base mismatches, improving RNA synthesis efficiency, and facilitating large-scale, high-quality RNA production.

Provided herein are compositions, kits, systems, and methods employing single-stranded end-preserving adaptors. Such single-stranded adaptors are attached to DNA duplex molecules while preserving original 5′ or 3′ single-strand protruding ends (e.g., present in cell-free DNA) by attaching such adapters to 3′ ends the DNA duplex molecules using a single strand ligase that has step 3 ligase activity, but not step 2 adenylyl transfer activity, and attaching such adapters to 5′ ends of the DNA duplex molecules using a ligase enzyme (e.g., a circligase), thereby forming loop-like structures on one or each end of the DNA duplex molecules. In further embodiments, the loop-like structures are cleaved (e.g., by an endonuclease) as the single-stranded adapters have a cleavable portion, thereby generating a two-part adapter on one or both ends of the DNA duplex molecules that preserves the initial 5′ or 3′ single-strand protruding ends, along with any methylation present.

The invention discloses a nucleic acid hybridization capture method and a kit, and the method comprises the following steps: constructing a hybridization reaction system, hybridizing the 5 '-phosphorylated probe to the nucleic acid sample wherein the 5'-portion and the 3 '-portion of the probe are complementary to a first region and a second region, which are not adjacent, in the nucleic acid sample, respectively, and a backbone portion connecting the 5'-portion and the 3 '-portion is not complementary to the nucleic acid sample; reacting the hybrid product under the action of nucleic acid polymerase and / or a polymerase active fragment, and nucleic acid ligase and / or a ligase active fragment to obtain a looping probe; and enriching the ring forming probe. Compared with the prior art, the method disclosed by the invention has the advantages of high specificity, high sensitivity, high flexibility, short process, convenience in operation, wide application scene and the like, and has a good application prospect.

The invention discloses a T4RNA ligase 2 mutant and a preparation method and application thereof, and relates to the field of biology and enzyme engineering, the T4RNA ligase 2 mutant provided by the invention has mutation at at least one site of 62th, 103th, 166th, 193th, 260th, 297th, 298th, 303th, 306th, 311th, 313th and 318th sites of a wild type T4Rnl2 sequence, compared with the wild type, the thermal stability of the mutant is obviously improved, and the T4RNA ligase 2 mutant can be used for preparing a T4RNA ligase 2 mutant. High ligase activity can be maintained at high temperatures such as 45 DEG C and 50 DEG C; in addition to being connected with a natural nucleic acid sequence, the mutant can also be connected with a non-natural nucleic acid with modification at high temperature. According to the mutant provided by the invention, ribonucleic acid can be synthesized at high temperature, so that by-products caused by base mismatch are effectively reduced, the RNA synthesis efficiency is improved, and large-scale high-quality RNA production is facilitated.

This invention discloses a T4 RNA ligase 2 mutant, its preparation method, and related applications, relating to the fields of biology and enzyme engineering. The T4 ligase 2 mutant provided by this invention has a mutation at at least one of the following positions in the wild-type T4 Rnl2 sequence: positions 90, 94, 133, 146, 160, 164, 177, 181, 186, 199, 201, 211, 215, 216, 252, 297, 307, and 330. The mutant provided by this invention exhibits significantly improved thermostability, maintaining high ligase activity even at high temperatures. This mutant can ligate not only native nucleic acid sequences but also efficiently ligate modified non-native nucleic acids. By performing RNA synthesis at high temperatures, byproducts caused by base mismatches can be effectively reduced, significantly improving RNA synthesis efficiency and product purity.

The invention discloses a low-temperature stress molecular regulator ATL31 / 6-CPK28, the plant molecular regulator comprises ATL31 and / or ATL6 and CPK28, ATL31 / 6 and a calcium signal component CPK28 in the plant molecular regulator form a dynamic interaction network: E3 ligase activity of ATL31 is activated under a low-temperature condition, E3 ligase is promoted to be combined with CPK28, ubiquitination degradation of the CPK28 is mediated, and the ATL31 / 6 and the CPK28 are activated under a low-temperature condition. Therefore, a negative feedback regulation loop induced by environmental stimulation is constructed. The invention provides important theoretical breakthrough and molecular targets for plant adversity biology research, has obvious scientific research originality, and also has basic, wide and important application prospects.

The invention discloses an LcfA enzyme mutant and application of the LcfA enzyme mutant in synthesis of Surfactin from bacillus subtilis, and belongs to the technical field of biology. According to the invention, bacillus subtilis LcfA enzyme is modified, Schrodinger molecular docking is combined with site-specific saturation mutation to establish a mutant library, Rosetta is used for screening the mutant library, target sites are searched, single-point mutation and combined mutation are carried out, the activity of long-chain fatty acid coenzyme A ligase is improved, and the synthesis efficiency of Surfactin is further improved. The obtained mutations comprise T217H, S332R, R452W, K538R and a combination of any two of T217H, S332R, R452W and K538R, fermentation verification shows that the yield of Surfactin is increased by 35.3% and reaches 9.2 g / L, a production strain with higher activity of long-chain fatty acid coenzyme A ligase and better fermentation performance in industrial production is obtained, and powerful support is provided for efficient production of Surfactin.

Provided herein are bifunctional compounds that bind tau protein and / or promote targeted ubiquitination for the degradation of tau protein. In particular, provided are compounds that can bind tau protein, a protein whose aggregation is implicated in a variety of neurodegenerative disease (e.g., tauopathies), and can promote its degradation by recruiting an E3 ubiquitin ligase (e.g., Cereblon), which can ubiquitinate tau protein, marking it for proteasomal degradation. Also provided are radiolabeled forms of the bifunctional compounds, pharmaceutical compositions comprising the bifunctional compounds, methods of detecting and / or diagnosing neurological disorders, methods of detecting and / or diagnosing pathological aggregation of tau protein (e.g., in the central nervous system), methods of treating and / or preventing neurological disorders, and methods of promoting the degradation of tau protein by E3 ubiquitin ligase activity in a subject by administering a compound or composition described herein.