13 results about "TEV protease" patented technology

The present invention relates to variants of TEV protease that have—compared to the wildtype enzyme—increased stability and catalytic activity as well as altered substrate specificity. The invention further relates to compositions comprising these variants as well as uses thereof and methods in which these variants are employed.

This invention discloses a β-glucosidase, its preparation method, and its applications, relating to the field of genetic engineering technology. The amino acid sequence of the β-glucosidase is shown in SEQ ID NO:1. The nucleotide sequence encoding this enzyme, optimized for codon bias in *E. coli*, is shown in SEQ ID NO:2. The constructed recombinant expression vector contains an IPTG inducible promoter, a ribosome binding site, a 6×His tag, a TEV protease recognition site, a flexible linker peptide, and the sequence shown in SEQ ID NO:2. The recombinant expression vector is transformed into *E. coli*, and after induction expression, high-density fermentation, and affinity purification, a highly soluble and active target enzyme is obtained. This enzyme can efficiently catalyze the conversion of ginsenoside Rb1 to rare ginsenoside CK, with a conversion rate exceeding 82%. This invention improves the soluble expression level and preparation efficiency of the target enzyme, making it suitable for industrial production.

The polypeptide membrane anchoring high-throughput screening method for GPCR is that the C terminal of GPCR is connected with tetracycline-controlled transcriptional activator (tTA) through tobacco etch virus protease substrate sequence (TEV-Substrate). When GPCR is combined with the membrane anchoring polypeptide, the TEV protease cuts the substrate to release tTA, activates the expression of luciferase reporter gene (Luc), and the cell expressing luciferase can detect the production amount of oxidized luciferin by using an enzyme label instrument, so that the accurate screening of the targeted polypeptide compound is realized. The screening method provided by the application can quickly screen the polypeptide combined with GPCR, is not limited to the screening of only a certain signal path in the traditional GPCR function detection, and can realize the high-throughput screening of the polypeptide and GPCR target by combining the luciferase reporter gene.

Disclosed is using a TEV protease (wild type or mutant) suited for removal from a reaction mixture, wherein the TEV protease displays a dual affinity tag including a chitin binding domain (CBD) tag and a histidine tag, preferably where the CBD tag precedes the N-terminus of the protease and the CBD tag is preceded by the histidine tag, which is preferably a 6-mer histidine tag; and optionally further including linkers, preferably Gly-Ser linkers, and more preferably a 6-mer Gly-Ser linker, between the tags. A linker, also preferably a Gly-Ser linker, can also follow the CBD tag and precede the protease portion. In certain embodiments, no such linkers are present and in other embodiments, only one such linker is present.

The invention belongs to the technical field of biology, and particularly relates to a recombinant human IL-15 protein preparation method based on an inclusion body renaturation process, which comprises the following steps: S1, constructing a recombinant vector 6H-tev-IL15 containing an IL-15 fusion protein coding sequence; s2, transforming the recombinant vector into an escherichia coli host cell BL21 (DE3) for induced expression to obtain an expression thallus; s3, crushing the expression thalli to obtain inclusion bodies, and washing the inclusion bodies; s4, dissolving the inclusion body by using a denatured buffer solution, carrying out primary purification through nickel affinity chromatography, collecting a protein eluent, and dialyzing to obtain denatured protein; s5, performing renaturation treatment on the denatured protein by using a renaturation buffer solution to obtain renatured protein; s6, cutting the renatured protein by using TEV protease, and carrying out nickel column affinity chromatography to obtain IL-15 protein flow-through liquid; and S7, collecting the IL-15 protein flow-through liquid, carrying out sephadex chromatography, and collecting the protein effluent at the peak to obtain the recombinant IL-15 protein.

According to some embodiments, the present disclosure relates to compositions, methods and kits for cleaving target proteins using cold-active variants of TEV protease.

The invention relates to TEV protease, in particular to a TEV protease variant and application thereof. The invention provides a TEV protease mutant, the nucleotide sequence of the TEV protease mutant is shown as SEQ.ID.2, and the amino acid sequence of the TEV protease mutant is shown as SEQ.ID.4. According to the invention, a gene of a TEV mutant is integrated into a pET28b vector and is transformed into a BL21 competent cell for expression, and protein is purified through IMAC (immobilized metal chromatography) and SEC (molecular exclusion chromatography). The TEV mutant shows higher expression quantity which is increased by 6 times compared with wild type TEV, meanwhile, the thermal stability and the activity of the TEV mutant are also improved, and part of protease is still remained after the TEV mutant is quickly heated at 95 DEG C, and the activity is still kept.

The application provides a purification method of foot-and-mouth disease virus capsid protein virus-like particles, and the purification method comprises the following steps: a fusion protein containing P1-2A is subjected to enzymolysis by 3C and TEV proteases to obtain an enzymolysis liquid; the enzymolysis liquid is subjected to nickel column purification by a nickel column to obtain foot-and-mouth disease virus capsid protein virus-like particle 5S protomers; and then foot-and-mouth disease virus capsid protein virus-like particles VLPs can be obtained by self-assembly. The purity of the foot-and-mouth disease virus capsid protein virus-like particles obtained by the purification method of the application is more than 90%, and the yield is more than 95%.

Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence-specificity. A limitation of TEV is its slow catalytic rate, which limits product generation and therefore signal output. Provided is a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is determined via proteolytic release of a membrane-anchored transcription factor, and access to TEV's cleavage site is temporally regulated using a photosensory LOV domain. By gradually decreasing light exposure time, faster variants of TEV were selected over multiple rounds of selection. The mutant TEV proteases and the directed evolution platform are useful in a wide range of biotechnology applications, such as FLARE and SPARK tools.

The invention discloses a eukaryotic protein expression and purification system as well as a construction method and application thereof, and belongs to the technical field of biology. The eukaryotic protein expression and purification system comprises an expression vector and a eukaryotic host cell, the expression vector contains a positioning peptide, an ELK16 aggregation tag and a connecting peptide. According to the system, protein enters an endoplasmic reticulum path through a positioning peptide; meanwhile, the ELK16 tag and the flexible connecting peptide are used for inducing to form an aggregate, so that the aggregate can stably exist in an endoplasmic reticulum environment of eukaryotic cells, cytoplasm degradation is avoided, and the protein yield and the purification efficiency are remarkably improved. Meanwhile, the system can release soluble and active target protein through TEV protease specific cleavage, and the purification process is simplified. Therefore, the system disclosed by the invention can systematically realize ELK16-mediated controllable aggregation and secretion coupling expression in the eukaryotic host, has the advantages of high yield, reusability and easiness in amplification, and has a good industrial application prospect.

This invention provides a fusion protein and its application in the preparation of Brazil sweetener. The fusion protein comprises a tag protein and a Brazil sweetener protein linked by a linker; wherein the linker comprises an amino acid sequence as shown in SEQ ID NO:1, and the amino acid sequence as shown in SEQ ID NO:1 is directly linked to the Brazil sweetener protein. This invention also provides a method for preparing the Brazil sweetener protein, comprising treating the fusion protein with a TEV protease and obtaining the Brazil sweetener protein. Large-scale expression of the fusion protein and the Brazil sweetener protein is achieved using an *E. coli* expression system; the resulting Brazil sweetener protein exhibits correct folding and a high sweetness.

The invention discloses a method for efficient recombinant expression of Brazzein sweet protein and application thereof, and belongs to the technical field of genetic engineering and recombinant microorganisms. The preparation method comprises the following steps: by taking pET-28a or pMAL-c6t as a carrier and Escherichia coli SHuffle T7 as a host, carrying out induced expression on an encoding gene of a sweet protein Brazzzein, and purifying, so as to obtain a fusion protein Brazzzein-His or His-MBP-Brazzzein; and the His-MBP-Brazzein is subjected to TEV protease cutting, and the cut His-MBP-Brazzein is purified to obtain the sweet protein mutant G-Brazzein. The sweetness of the obtained sweet protein mutant G-Bragg zein exceeds 1500 times of that of cane sugar, the sweet protein mutant G-Bragg zein can still taste obvious sweetness under the concentration of 6.6 mu g / mL, and the sweetness is pure; the sweetness of the Bragg zein-His is 467 times that of cane sugar, the lasting time of the sweetness is as long as 12s, and additional enzyme digestion is not needed in the preparation process. The obtained brazzein sweet protein can be used for improving coffee fragrance and reducing the sugar consumption.

This invention provides TEV protease mutants and their applications, relating to the field of synthetic biology. By introducing mutation sites into the TEV protease, this invention constructs different TEV protease mutants and discovers beneficial mutation sites that can directly increase expression levels. This provides new genetic engineering sites and a research foundation for the study of TEV protease expression systems and yield improvement. This invention uses a highly efficient *E. coli* protein expression system to express the TEV protease mutants. These TEV protease mutants not only possess the functional activity and specificity of the natural enzyme but also avoid intramolecular cleavage, maintain the activity and stability of the protease, and exhibit high soluble expression levels of TEV protein, effectively saving downstream purification costs. This provides a theoretical basis for the large-scale production of TEV protease.