120 results about "Intein" patented technology

To provide constructs, vectors, relative host cells and pharmaceutical compositions which allow an effective gene therapy, in particular of genes larger than 5Kb.SOLUTION: The present invention implements cellular large protein reconstitution by providing to a target cell two or more fragments of large protein fused to split inteins to promote intein-mediated trans-splicing and reconstitute the functional protein.SELECTED DRAWING: None

The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant collagen based on engineering bacterium expression and a preparation method thereof, and the recombinant collagen is formed by connecting repetitive units derived from human III type collagen in series for three times; the invention also discloses a base sequence of the coding gene of the recombinant collagen, efficient expression and accurate folding of target protein are realized through genetic modification of engineering bacteria, a light-operated dynamic folding process and an orthogonal purification strategy, and cascade purification of a His6-SUMO tag and an intein-CBD module is utilized to obtain the recombinant collagen. The product purity and the native conformation retention rate are remarkably improved, and the technical problems that hydroxyproline depends on exogenous addition, the folding controllability is poor and the purification efficiency is low in a traditional process are solved.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

The invention discloses a split intein and application thereof, the split intein comprises two independent peptide fragments: (a) an N-terminal fragment, the amino acid sequence of which is as shown in SEQ ID NO.1; (b) a C-terminal fragment or a variant thereof, the amino acid sequence of the C-terminal fragment is as shown in SEQ ID NO.2, and the amino acid sequence of the variant is as shown in SEQ ID NO.3 or SEQ ID NO.4. The split intein has splicing activity and excellent splicing rate, the application of the split intein can be widened, the number of amino acids of the C-terminal fragment or the variant thereof is small, and the split intein has remarkable advantages in protein purification, solid-phase synthesis and the like.

The invention relates to a first fusion protein comprising N-terminally an N-terminal fragment of otoferlin and C-terminally an N-terminal fragment of an intein, and a second fusion protein comprising N-terminally a C-terminal fragment of an intein and C-terminally a C-terminal fragment of intein.

The invention relates to the technical field of molecular biology and protein engineering, in particular to a lactic acid bacteria antibacterial peptide LABA4 multi-copy expression method and application. The invention aims to provide a prokaryotic expression method of an antibacterial peptide LAB4 tandem gene based on a self-assembled peptide and application, so that the problems of toxicity to a host, low expression quantity and non-ideal product activity in a large intestine expression system are solved, the antibacterial peptide is efficiently expressed in escherichia coli, and the antibacterial activity is effectively improved. Particularly, the characteristics of specific cutting and no amino acid residue of enterokinase are utilized, modified recombinant enterokinase is added between LAB4 monomers to serve as an interval restriction enzyme cutting site, a gene shuffling technology is utilized to design and construct three-six copy tandem recombinant plasmids of LAB4, the self-cleavage characteristic of intein is utilized, and the recombinant enterokinase recombinant plasmids of LAB4 can be used for preparing the recombinant enterokinase recombinant plasmids of LAB4, so that the recombinant enterokinase recombinant plasmids of LAB4 can be used for preparing the recombinant enterokinase recombinant plasmids of LAB4. An optimized cspA cold shock promoter in a pCold I vector is adopted, and high-efficiency expression of tandem LAB4 in an escherichia coli expression system is realized through 15 DEG C low-temperature induction precise regulation and control.

Described herein are fusion proteins comprising intein polypeptides and methods of using these proteins. The fusion proteins described herein may include a Cas12a polypeptide and an intein polypeptide or a polypeptide of interest (e.g., a reverse transcriptase polypeptide) and an intein polypeptide. Also described herein are compositions and systems for modifying or editing a target nucleic acid.

The present invention relates to a composition and a method for expressing an OTOF gene by a dual-vector system. Specifically provided are a fusion polypeptide, comprising an otoferlin protein portion, and an intein system portion. The gene encoding OTOF is transferred into the cell by means of a recombinant AAV dual-vector system.

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.

The present application is directed to multiplex intein-based methods and compositions for the generation engineered cells expressing modular polypeptides, for example CARs and CCRs.

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.

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 thus 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 thus the second addition component of the second modification protein is linked to the second monomeric protein.

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.

This invention discloses an intima-in-the-liquid peptide and its application in the biological preparation of tetrapeptide-7. The amino acid sequence of the Cth TerA intima-in-the-liquid peptide variant is shown in SEQ ID NO:3. In this invention, a highly efficient and stable recombinant expression vector for tetrapeptide-7 is prepared by combining the Cth TerA intima-in-the-liquid peptide variant with tetrapeptide-7 to form a fusion protein. After being introduced into a vector plasmid, an engineered bacterial strain is obtained. High-purity, unmodified tetrapeptide-7 can be obtained through induced expression. This method is simple, rapid, produces no toxic byproducts, and yields large quantities, making it suitable for large-scale industrial production of tetrapeptide-7. This results in high-quality tetrapeptide-7 with significant market value.

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 present disclosure is generally related to filamentous fungal strains comprising genetic modifications that give rise to recombinant cells having altered phenotypes, wherein such recombinant cells are particularly well-suited for growth in submerged cultures (e.g., for the large-scale production of proteins of interest). Certain embodiments of the disclosure are related to, inter alia, compositions and methods for designing, constructing, testing, screening, growing, fermenting, and the like recombinant filamentous fungal strains expressing intein-modified fitness proteins.

The invention relates to an efficient preparation and activation method of natural protein glutaminase, and belongs to the technical field of biology. The invention provides a method for preparing and activating natural protein glutaminase, which comprises the following steps of: expressing fusion protein consisting of super-folded green fluorescent protein, a protein glutaminase propeptide region, intein and a protein glutaminase mature region in bacillus subtilis; extracellular expression of fusion protein is guided by super-folded green fluorescent protein, the fusion protein is spontaneously cut by intein Mxe GyrA, activated protein glutaminase is obtained, the sequence of the activated protein glutaminase is completely consistent with that of natural protein glutaminase, the highest enzyme activity reaches 52.3 U / mL, and the activity of the protein glutaminase is remarkably improved. The problem that the mature enzyme sequence is changed due to poor protease activation specificity in the prior art is solved, a brand new thought and tool are provided for efficient preparation and activation of protein glutaminase, and the method has wide application prospects.

The present invention relates to a method for producing botulinum toxin in a recombinant manner by using a soluble partner and intein. In the present invention, it was confirmed that HC having low solubility can be expressed in a soluble form by attaching a soluble partner. It was also confirmed that a soluble partner is generally a protein having a very high molecular weight, and when intein is used in the present invention, the soluble partner can be easily removed.

In order to develop a therapeutic strategy to rescue ABCA3 deficiency caused by a splicing mutation of ABCA3, we have designed an experimental scheme using CRISPR / Cas9 to delete a loxP site located at an intronic region of ABCA3 flox mice (Sftpc-Cre;Abca3flox / flox). The deletion of this loxP site extended survival of the mice by only a week, probably due to insufficient re-ligation efficiency after the CRISPR / Cas9 deletion. An alternative approach to rescuing ABCA3 deficiency caused by mutations in ABCA3, including coding or non-coding (e.g., intronic) mutations, is to deliver intact ABCA3 into lung alveolar type II cells using AAV (e.g., AAV6.2FF), a clinically approved delivery system. Due to the large size of ABCA3 (1704 amino acids), it is technically impossible to package full length ABCA3 cDNA into an AAV virus. However, a recent technological advance using intein makes it possible to split ABCA3 cDNA into two fragments for separate AAV delivery to the cells wherein the translated ABCA3 protein fragments would recombine into full length ABCA3 protein.

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 invention discloses a mitochondrial base editor with extremely low cell nucleus off-target. MTS in the editing system is selected from a 2xMTS form of any one of seven mitochondrial targeting positioning signals, UGI in the editing system is in an Inteins splitting form or a Splitts splitting form, a 2xMTS-UGI (Inteins) or 2xMTS-UGI (Splitts) skeleton carrier formed by the MTS and the UGI realizes C / G-T / A editing of a target position on mitochondrial DNA, and the off-target value of a cell nucleus is extremely low. On the basis of the 2xMTS-UGI (Inteins) or 2xMTS-UGI (Splitts) skeleton vector, the double-stranded deamination protein inhibitory enzyme is jointly expressed, C / G to T / A editing of the target position on the mitochondrial DNA is also realized, the off-target value of the cell nucleus is as low as the background level, and a safer tool is provided for the gene editing technology.

Provided herein are synthetic polypeptides including trans-splicing inteins, functional fragments thereof, and polynucleotides encoding the same for use in systems, compositions, kits, and methods for delivering one or more polynucleotides (e.g., polynucleotides encoding a split polypeptide) to a cell using a vector (e.g., a viral vector, such as an adeno-associated virus vector) having limited packaging capacity.

The invention relates to a method for producing botulinum toxin by utilizing a solubilizing partner and GP41.1 intein, in particular to a method for producing the botulinum toxin by utilizing the solubilizing partner and the GP41.1 intein in a recombination manner. The present invention confirms that the receptor binding domain (HC) of the heavy chain of botulinum toxin, which is less soluble, can be expressed in a soluble form in Escherichia coli by adding a solubilizing partner (Soluble Partner). Moreover, the solubilizing partner is usually a protein having a very high molecular weight, but the present invention confirms that the solubilizing partner can be easily removed using the GP41.1 intein.

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 present invention relates to protein purification, mainly in the field of chromatography. More specifically, the present invention relates to affinity chromatography using a split intein system with an improved C-intein tag and an N-intein ligand, wherein the target protein can be purified as a tag-free end product with a native N-terminus.

To provide a composition and a method for helping achievement of delivery of CRISPR / Cas9 component to a cell.SOLUTION: Provided are a composition and a method for delivering first and second polynucleotide severally encoding fragments of A→G base editor fusion protein containing one or more deaminase (e.g., adenosine deaminase) and nCas9, where the first polynucleotide encodes an N-terminal fragment of nCas9 fused to intein -N of a divided intein pair, and the second polynucleotide encodes a C-terminal fragment of nCas9 fused to intein -C of a divided intein pair. A method for delivering (e.g., AAV delivery) these fragments together with sgRNA cell is also provided, where these fragments are connected together by a divided intein system, by which a functional base editing system is reconstituted in a cell.SELECTED DRAWING: Figure 1

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.