45 results about "Intein" patented technology

The present disclosure relates to enzymes, compositions and methods for performing conditional homologous recombination of a targeted DNA molecule or genome by using modified proteins comprising nucleic acid DNA binding proteins with protease-conditional recombinase (hereinafter “ProRec”) activity. Namely a new cre recombinase pro enzyme, compositions / kit and sensors comprising the cre recombinase pro enzyme of the present disclosure. Namely, a cre recombinase pro-enzyme, for identifying and quantifying proteolytic activity in a disease or an infection, comprising intein mediated circularization, a flip-excision cassette comprising an output reporter and / or effector protein; a linker sequence cleavable by a protease.

Provided is a method for producing a heterodimeric protein such as a bispecific antibody with which a heterodimeric protein composed of only domains having natural amino acid sequences can also be produced. A production method of the present invention is a method for producing a heterodimeric protein. The production method includes a production step of producing a heterodimeric protein by reacting a dimeric protein having a reaction tag with a modification protein for modifying the dimeric protein. The dimeric protein having a reaction tag includes a first monomeric protein and a second monomeric protein. The first monomeric protein includes a first reaction tag and a first dimer formation domain capable of forming a dimer in this order. The first reaction tag includes a binding tag and a first C intein. The second monomeric protein includes a second reaction tag and a second dimer formation domain capable of forming a dimer together with the first dimer formation domain in this order. The second reaction tag includes a binding partner capable of binding to the binding tag, and a second C intein. The modification protein includes a first modification protein and a second modification protein. The first modification protein includes a first N intein capable of reacting with the first C intein, and a first addition component to be added to the first monomeric protein. The second modification protein includes a second N intein capable of reacting with the second C intein, and a second addition component to be added to the second monomeric protein. The first addition component and the second addition component are different addition components. The first monomeric protein and the second monomeric protein form a dimer. In the production step, the first N intein of the first modification protein reacts with the first C intein of the first monomeric protein, and then the first addition component of the first modification protein is linked to the first monomeric protein, and the second N intein of the second modification protein reacts with the second C intein of the second monomeric protein, and then the second addition component of the second modification protein is linked to the second monomeric protein.

This invention discloses an inteptide Ssp tau, characterized by its amino acid sequence as shown in SEQ ID No. 2. It also discloses its encoding gene and its application in the preparation of tetrapeptide-5. Furthermore, it discloses the Ssp tau-tetrapeptide-5 recombinant protein, the corresponding encoding gene, the expression vector, and the expression host bacteria. This invention discloses a novel inteptide Ssp tau and an inteptide Ssp tau-mediated tetrapeptide-5 expression and purification technique. Ssp tau and tetrapeptide-5 are combined to form a fusion protein, and the fusion protein gene sequence is constructed into PET-28a(+) to obtain a recombinant expression vector for tetrapeptide-5. Through simple steps such as induced expression, cell disruption, ultrafiltration, inteptide autocleavage, secondary ultrafiltration, and rotary lyophilization, high-purity tetrapeptide-5 can be obtained efficiently. This biosynthetic method is suitable for the industrial production of tetrapeptide-5 and has significant market value.

The present invention is directed to lipid nanoparticles, compositions, formulations and systems containing lipid-based nanoparticles and methods of treating diseases or conditions with said lipid nanoparticles, compositions, formulations and systems thereof. The present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding an N-terminal portion of a polypeptide of interest and a nucleotide sequence encoding an N-terminal intein, and (b) a targeting molecule displayed on the outer surface of the lipid nanoparticle. The present disclosure provides a lipid nanoparticle comprising (a) a nucleic acid comprising a nucleotide sequence encoding a C-terminal portion of a polypeptide of interest and encoding a C-terminal intein, and (b) a targeting molecule displayed on the outer surface of the lipid nanoparticle.

The invention relates to the combination of a cell-selective promoter to drive the N-terminal part of the transgene and a compact ubiquitous promoter with similar expression levels to drive the C-terminal part of the transgene (or vice versa) for dual AAV intein-mediated delivery to obtain cell-selective, efficient transgene expression.

The invention provides compositions and methods for delivering first and second polynucleotides each encoding a fragment of an A-to-G Base Editor fusion protein comprising one or more deaminases (e.g., adenosine deaminases) and nCas9, wherein the first polynucleotide encodes an N-terminal fragment of nCas9 fused to an intein-N of a split intein pair and the second polynucleotide encodes a C-terminal fragment of nCas9 fused to an intein-C of a split intein pair, and methods for delivering these fragments together with an sgRNA to a cell (e.g., AAV delivery), where the fragments are spliced together by a split intein system, thereby reconstituting a functional base editing system in the cell.

The present invention relates to an expression system and a method ensuring constant-level concentration of an output. The invention also relates to a cell comprising the expression system.

The invention discloses an expression cassette combination and application thereof. The expression cassette combination comprises a first expression cassette and a second expression cassette, the first expression cassette can express a fusion protein of an N-terminal truncation of ABCA4 protein and an intein N terminal, the second expression cassette can express a fusion protein of a C-terminal truncation of ABCA4 protein and an intein C terminal, and when expressed, the fusion protein of the C-terminal truncation of ABCA4 protein and the intein C terminal is separated from the fusion protein of the C-terminal truncation of ABCA4 protein. The N-terminal truncation of the ABCA4 protein expressed by the first expression cassette and the C-terminal truncation of the ABCA4 protein expressed by the second expression cassette can form a complete full-length ABCA4 protein through the shearing function of the N terminal and the C terminal of intein.

The invention provides an AsCas12f gene segmentation expression mediated gene editing method. The AsCas12f gene segmentation expression mediated gene editing method is provided. The key point is that the AsCas12f gene is divided into two parts, the two parts are respectively cloned to expression vectors, and gene editing is realized after the two vectors are co-expressed. Preferably, the front half part of the AsCas12f comprises REC, WED and Linker sequences, the rear half part of the AsCas12f comprises Linker, RuvC and TNB sequences, a gRNA sequence and a target gene sequence, the two parts contain Linker overlapping sequences, a 5'end sequence of intein is added to the 3'end of the front half part, a 3 'end sequence of the intein is added to the 5' end of the rear half part, the two parts are expressed by utilizing plant virus vectors TRV (tobacco brittle virus) and PVX (potato virus X) respectively, and the AsCas12f is expressed by utilizing plant virus vectors. And gene editing of the target gene is realized.

The present disclosure is directed to systems and methods for synthesizing a spidroin. In some embodiments, the methods comprise synthesizing a monomer in vivo in a heterologous host, the monomer comprising an N-terminus IntC domain and a C-terminus IntN domain, and post-translationally polymerizing the synthesized monomer via in vitro split-intein mediated polymerization.

The present disclosure relates to the use of split inteins for expressing Myosin-VIIa protein encoded by MYO7A gene in a subject in need thereof for gene therapy in particular for the treatment of MYO7A associated disease, preferably Usher syndrome type 1B.

Embodiments of the present disclosure are directed to a plurality of nucleotide sequences encoding a first intein fused to at least a portion of a first transcription activator-like effector (TALE), a second nucleotide sequence encoding the first intein fused to at least a portion of a second TALE, and a third nucleotide sequence encoding a second intein fused to at least a portion of a rare-cutting nuclease.

This invention belongs to the field of biopharmaceutical technology, specifically relating to a modular protein delivery system for targeted intracellular delivery of sustained-activation cGAS protein and its applications. The modular protein delivery system comprises a first functional module and a second functional module: the first and second functional modules are connected via a fragmentation-type intron or a Cage-Colockr system; the first functional module includes the cholera exotoxin B subunit or a functionally conserved variant thereof; the second functional module includes a sustained-activation cGAS mutant protein or a lysosomal degradation-resistant derivative thereof. The modular protein delivery system provided by this invention can be used to prepare immunotherapeutic drugs for the prevention or treatment of tumors, exhibiting advantages such as strong targeting and significant immune activation effects.

The present invention relates to endoproteins and uses thereof. The present invention relates to constructs, vectors, related host cells and pharmaceutical compositions that allow for effective gene therapy, in particular for genes greater than 5 Kb.

We describe an artificial expression construct for rescuing voltage-gated sodium channel function by intein-mediated reconstitution of voltage-gated sodium channel alpha subunit 1 (Nav1.1), a protein encoded by SCN1A. Rescued voltage-gated sodium channel function can be utilized to treat disorders such as epilepsy, and more specifically, Dravet syndrome.

This invention discloses a method and its application for predicting the trans-splicing activity of fragmented inpeptides. The method takes the amino acid sequence of the fragmented inpeptide as input, extracts 1280-dimensional residue-level sequence embedding features using the ESM-2 protein language model, and uses AlphaFold2 for high-precision three-dimensional structure prediction to obtain the spatial coordinates of the residues. A graph structure is constructed with residues as nodes and residue pairs with a Cα atomic distance of less than or equal to 8 Å as edges. Node features include sequence embedding and chain affiliation information, while edge features include Cα distance and cross-chain connection identifiers. The graph structure is input into the DeepIntein deep learning model based on a graph attention network, and the splicing activity prediction probability is output after multi-layer graph attention convolution, global pooling, and fully connected layers. The prediction throughput of this invention is more than three orders of magnitude higher than that of the traditional Western blotting method, breaking through the limitations of homology sequence exploration space.

Embodiments of the present disclosure are directed to a plurality of nucleotide sequences encoding a first intein fused to at least a portion of a first transcription activator-like effector (TALE), a second nucleotide sequence encoding the first intein fused to at least a portion of a second TALE, and a third nucleotide sequence encoding a second intein fused to at least a portion of a rare-cutting nuclease.

The application provides an AsCas12f gene segment expression mediated gene editing method. The gist is that the AsCas12f gene is divided into two parts, which are respectively cloned into expression vectors, and gene editing is realized after co-expression of the two vectors. Preferably, the first half of the AsCas12f gene comprises REC, WED and Linker sequences, the second half comprises Linker, RuvC and TNB sequences, gRNA sequences and target gene sequences, the two parts contain overlapping Linker sequences, the 3' end of the first half is added with the 5' end sequence of an Intein, the 5' end of the second half is added with the 3' end sequence of the Intein, the two parts are respectively expressed by plant virus vectors TRV (tobacco rattle virus) and PVX (potato virus X), and gene editing on the target gene is realized.

The invention discloses an intein-based short peptide screening platform for disturbing a liquid-liquid phase separation state, which is characterized by comprising a first expression construct, a second expression construct and a third expression construct, the targeting protein sequentially comprises a disordered region of a phase separation protein, a first fluorescent protein and an N-terminal fragment of intein from an N terminal to a C terminal, the second expression construct comprises a second nucleic acid sequence for coding a targeted binding protein, and the targeted binding protein sequentially comprises a C-terminal fragment of intein, a second fluorescent protein and a to-be-screened oligopeptide from the N terminal to the C terminal; wherein when the first expression construct and the second expression construct are co-expressed in a host cell, protein splicing is carried out on an N-terminal fragment and a C-terminal fragment of the intein, and the oligopeptide to be screened is delivered to a liquid-liquid phase separation aggregate formed by a disordered region of the phase separation protein, therefore, the active oligopeptide capable of disturbing the liquid-liquid phase separation aggregate is screened out.

This invention relates to a genetically engineered bacterial outer membrane vesicle and its preparation method. By using in-cell peptides to attach exogenous proteins to the surface of OMVs via peptide bonds, a variety of desired antigens can be conveniently delivered. The reaction conditions are mild, less affected by antigen size, and unlike the SpyCatcher / SpyTag system, the in-cell peptides do not remain in the mature protein after attaching the exogenous protein to the OMV surface. This invention can serve as a universal tool for simultaneously or separately displaying different antigens on the surface of OMVs.

The present invention relates to a dual vector system for expressing Otoferlin protein, comprising a first nucleic acid vector and a second nucleic acid vector wherein the first nucleic acid vector comprises a first nucleotide sequence; the second nucleic acid vector comprises a second nucleotide sequence; the first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences; the second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences; the expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of Otoferlin, an N-terminal coding sequence of intein and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of intein, a C-terminal coding sequence of Otoferlin and polyA, wherein the first amino acid of the C-terminal coding sequence of Otoferlin is mutated into serine (S), threonine (T) or cysteine (C). The invention also relates to a packaging carrier system of the adeno-associated virus, a packaging method of the adeno-associated virus and the adeno-associated virus obtained by the method. The dual vector system or adeno-associated virus for expressing the Otoferlin protein of the present invention can be used in gene therapy, particularly in the treatment of hearing loss, such as in the treatment of Oof gene mutation associated autosomal recessive hereditary hearing loss 9 (DFNB9).

Described in several example embodiments herein are engineered split intein polypeptides and systems thereof. Also described in several example embodiments, herein are methods of using the engineered split intein polypeptides and systems thereof, such as to catalyze a bioconjugation reaction.

The present application relates to recombinant expression of proteins within cells, including expression of therapeutic molecules. In some aspects, the present application relates to an intein-based split biomarker selection system allows using a single selection pressure for the incorporation of multiple transgenic fragments simultaneously.

The present invention relates to fusion proteins and methods of using the same. Specifically, invention relates to fusion proteins comprising an intein and a DNA polymerase, and methods of using the same for DNA synthesis.

A dual-vector system expressing Otoferlin protein, comprising a first nucleic acid vector and a second nucleic acid vector, the first nucleic acid vector comprising a first nucleotide sequence, and the second nucleic acid vector comprising a second nucleotide sequence; the first nucleotide sequence comprises an expression cassette inserted between two first ITR sequences; the second nucleotide sequence comprises an expression cassette inserted between two second ITR sequences; the expression cassette of the first nucleotide sequence comprises a promoter, an N-terminal coding sequence of Otoferlin, an N-terminal coding sequence of an intein, and polyA; the expression cassette of the second nucleotide sequence comprises a promoter, a C-terminal coding sequence of an intein, a C-terminal coding sequence of Otoferlin, and polyA, the first amino acid of the C-terminal coding sequence of Otoferlin being mutated to serine (S), threonine (T) or cysteine (C). An adeno-associated virus packaging vector system, an adeno-associated virus packaging method, and an adeno-associated virus obtained therefrom are also provided. The dual-vector system or adeno-associated virus for expressing Otoferlin protein can be used for gene therapy, particularly for treating hearing loss, such as for treating Otof gene mutation-related autosomal recessive nonsyndromic deafness 9 (DFNB9).

The invention discloses a method for enhancing recombinant protein expression and phase separation, which comprises the following steps: performing fusion expression on a phase separation peptide, a connecting peptide, intein and a target protein in a host cell, crushing the host cell, centrifuging, and taking precipitate, thereby obtaining a fusion protein inclusion body. The phase separation peptide and the adaptive connecting peptide thereof not only can significantly improve expression of a recombinant protein inclusion body, but also can enhance the aggregation effect of the recombinant protein, reduce the production cost of the recombinant protein and improve the purity of the target protein, are suitable for production of various recombinant human-derived proteins, and have wide application prospects. The method lays a foundation for efficient expression and separation of various recombinant protein inclusion bodies, and has a good commercial application prospect.

The invention discloses a recombinant expression and in-vitro splicing integrated preparation method and application of spider silk protein, and belongs to the technical field of biology. Coding genes of NT, Rep or CT regions of spider silk protein from at least two different spider species are fused, a fusion gene sequence is optimized by pichia pastoris codon and then introduced into an Intein tag, and the spider silk protein is obtained. Dividing the full-length protein into an N-terminal fragment and a C-terminal fragment; respectively cloning the N-terminal fragment and the Intein-N structural domain as well as the C-terminal fragment and the Intein-C structural domain into a pichia pastoris secreting type expression vector pPICZalpha; respectively electrically transforming the two obtained vectors into pichia pastoris SMD1168 for induced expression, and collecting supernatant; sequentially carrying out Ni-NTA affinity chromatography and ion exchange chromatography on the supernatant to obtain a purified N-terminal protein fragment and a purified C-terminal protein fragment; mixing the spider silk protein and the spider silk protein, forming full-length spider silk protein through Intein-mediated trans-splicing, and purifying through size exclusion chromatography. The method provided by the invention can realize high-efficiency and high-quality production of spider silk protein.

The present invention relates to a method of identifying whether an interaction domain capable of nucleating self-assembly of a plurality of fusion proteins comprising said interaction domain and capsid-forming proteins into synthetic transfer vehicles (STVs) provides for RNA transfer efficiency of said STVs from a sender to a receiver cell, comprising: (A) providing a mammalian sender cell; (B) providing a mammalian receiver cell; (C) bringing supernatant from said sender cell comprising the population of STVs into contact with said receiver cell, thereby allowing fusion of said population of STVs with the cell membrane of said reporter cell and delivery of first polynucleotides into the reporter cell; and (D) determining the signal obtained from the reporter cell, said signal being produced by the functional reporter system through protein-fragment complementation of the first and second member of said first split reporter system, said protein-fragment complementation being mediated by protein splicing through a functional intein forming by self-association of the first and second entity of the split intein, said self-association being mediated by the interaction of the protein- protein interaction domains encoded by the first and fourth polynucleotide, respectively; thereby identifying an interaction domain which provides for RNA transfer efficiency of STVs from a sender to a receiver. The present invention further relates to a non-naturally occurring interaction domain obtainable by the method of the present invention.

The present disclosure relates to methods of protein purification by attaching an intein-C fragment to a target protein, passing a sample containing the intein-C tagged protein over a chromatographic resin carrying an intein-N fragment so as to create an intein-N intein-C complex, releasing the target protein from the intein-C fragment, and regenerating the column under conditions that disrupt the intein-N intein-C complex while preserving column functionality for multiple reuses.

The present disclosure relates to the use of split inteins for expressing centrosomal protein 290 (CEP290) protein encoded by CEP290 gene in a subject in need thereof for gene therapy in particular for the treatment of CEP290-associated disease, preferably Leber congenital amaurosis.