36 results about "TEV protease" patented technology

Polynucleotide constructs that express an engineered foot-and-mouth disease (FMDV) P1 precursor protein and a non-FMDV TEV protease and methods for safe and efficient recombinant production of FMDV antigens and immunogens. Recombinant production of FMDV antigens avoids the need to culture highly-infectious FMDV, while conventional culture methods for producing FMDV antigens rely on the native FMDV 3C protease which exerts toxic effects on host cells. The inventors have developed a new system that efficiently and safely processes FMDV P1 precursor without the FMDV 3C protease, thus avoiding the toxic effects associated with use of the 3C protease. The invention is also directed to the FMDV antigens and virus-like particles produced by this system as well as to FMDV vaccines, diagnostics and other biologics.

The invention belongs to the technical field of gene engineering and particularly relates to a fusion expression method of AEP cyclase in Escherichia coli, a method for identifying cyclization capacity of the AEP cyclase and application of the APE cyclase. Fusion expression is performed in the Escherichia coli by different fusion labels, then, the AEP cyclase with higher expression is obtained after the fusion labels are removed, and the expression of the AEP cyclase is 0.36 plus/minus 0.05 mg/mL, which is higher than 1.8 mg/L reported in original literature. The new method is adopted to verify the cyclization capacity of the AEP cyclase, protein which can be better observed in SDS-PAGE and has cyclization probability is selected by constructing a serial substrate, a serial substrate containing a substrate sequence of TEV protease is constructed, whether cyclization is performed according to change of the size of the substrate or the number of stripes after protease cutting, and the method is simple to operate, convenient and high in practicability.

The invention provides a TEV protease mutant, a gene, a biomaterial, a preparation method, a reagent, or a reagent kit and application, and relates to the technical field of genetic engineering. Compared with a protease mutant having an amino acid sequence shown as SEQ ID NO.1, the TEV protease mutant is mutated into at least seven amino acid residues, for example 17th site is mutated into S fromT, the 56th site is mutated into V from L, the 68th site is mutated into D from N, the 77th site is mutated into V from I, the 135th site is mutated into G from S, the 219th site is mutated into V from S, and the 233th site is mutated into H from Q. The TEV protease mutant has favorable enzymatic activity, stability and specificity. The gene, the biomaterial and the preparation method of the TEV protease mutant, or the reagent or the reagent kit containing the TEV protease mutant, the application of the technical scheme, and the TEV protease mutant are all based on the same invention conception.

The invention provides a method for massively producing human thymosin with low cost. The method is used for implementing restriction enzyme digestion to obtain a fusion gene sequence with a structure as follows: A-X-C, wherein A is a nucleotide sequence of site (1-150)-(1-372) amino acids at the N- end of coded mature human serum albumin; X is the nucleotide sequence of connecting peptide with enterokinase or tobacco etch virus (TEV) protease cutting site contained in a code; and C is a human thymosin gene. The method comprises steps as follows: connecting the fusion gene sequence to an expression carrier; transforming and introducing the expression carrier into saccharomycetes; carrying out induction expression to obtain soluble human serum albumin-thymosin fusion protein; then adding the TEV protease for cutting; and separating and purifying to obtain the recombined human thymosin. The human thymosin production method provided by the invention has the advantages that consistency of quality of products can be ensured, no limitation is generated from sources of raw materials, cost is low, an expression index is high, and mass production can be achieved and the like.

Provided are methods for protein engineering, such as engineering proteases or kinases. The methods may utilize yeast display and/or ER sequestration of proteins or substrates. In some aspects, TEV proteases with altered substrate specificity, potency, and/or efficiency are provided.

The invention discloses a method for producing recombinant TEVprotease in escherichia coli. The method comprises the following steps: S1) construction of PET28aTEV protease expression plasmid; S2) inducing expression and identification of TEV protease; and S3) purification and identification of TEV protease. The invention adopts the escherichia coli expression system to realize the expression of recombinant TEV protease in escherichia coli by designing an expression vector and then adopts affinity chromatography and Superdex 75 gel filtration chromatography to purify the target protease, thusgreatly shortening the purification process and time of the target protease, improving the purity, yield and activity of the protease, saving experimental steps and reducing cost. The method providedby the invention lays a foundation for the development and production of other protein tool enzymes.

The invention relates to a method of separating a target protein by shearing a fusion protein by escherichia coli intracellular protease. Used host bacteria is obtained by integrating a TEV (tobacco etch virus) protease gene driven by a strong promoter T7 to escherichia coli expression host bacteria BL21(DE3) by virtue of a recombinant engineering method. An expression vector is obtained by cloning maltose-binding protein and stuffer fragment to the expression vector pET30(+). A target gene not containing a terminator replaces the stuff segment to obtain a target gene fusion expression vector, and the tail end of the fusion protein contains 6 histidines of the vector. After the expression vector is transformed to the host bacteria, under induction of isopropyl-beta-D-thiogalactoside, the fusion protein and the TEV protease based on a chromosome are expressed at the same time; the TEV is acted on an enzyme cutting site between the maltose-binding protein and the target protein for separating the target protein. According to the intracellular shearing method, steps of fusion protein separating, TEV enzyme digesting, and the like are omitted.

The invention builds up a novel method for preparing recombinant exenatide or derivative thereof. According to the method, hirudin (molecular weight is 7Kd) serves as a fusion partner, exenatide or derivative thereof is spiced on the downstream of the hirudin for fusion expression, a connecting peptide is arranged between the hirudin fusion partner and the exenatide or derivative thereof and comprises a TEV protease (tobacco etching virus protease) identification cutting sequence ENLYFQH. The exenatide or derivative thereof with a natural N-terminal and biological activity is released through TEV enzyme digestion after fusion protein expression. The novel method has other advantages that (1) the hirudin fusion partner is small, so the ratio of the exenatide or derivative thereof in fusion protein is effectively increased; (2) hirudin fusion protein has anticoagulation activity, thus facilitating real-time detection and tracing; (3) fusion protein can be adsorbed by virtue of cheap macroporpous resin; and (4) hirudin fusion partner can enhance stability of exenatide or derivative thereof.

The present invention relates to a method for screening a protease variant and the obtained protease variant, and the protease variant has a special performance. The method for screening the protease variant comprises the following steps: (1) constructing a protease random mutation library, optimizing a protease random mutation virus library, and optimizing a protease random mutation phage library; (2) for the protease random mutation library, optimizing the protease random mutation virus library, and optimizing the protease random mutation phage library and screening a protease variant having weak protease enzyme-cutting activity in a host or a similar condition and having protease enzyme-cutting activity in an in vitro denaturation condition; and (3) the step of optionally characterizing the protease variant, wherein the protease optimizes TEV protease or enterokinase. The protease variant in the present invention facilitates industrial production of protein.

The invention discloses an efficient-expression recombinant TEV enzyme with high activity and stability as well as a preparation method, a determination method and application of the recombinant TEV enzyme. The recombinant TEV enzyme comprises an amino acid sequence for coding TEV protease or a mutation sequence of the amino acid sequence, a CL7 tag is fused at the N end of the amino acid sequence for coding the TEV protease or the mutation sequence of the amino acid sequence, a polyarginine tag is fused at the C end of the amino acid sequence for coding the TEV protease or the mutant sequence of the amino acid sequence. Compared with existing recombinant TEV protease, the recombinant TEV protease not only can overcome the defect that the protein stability and activity are reduced due to the fact that the wild type TEV protease shears own specific sites in the expression process, but also has higher protein yield, better protein stability and higher activity, the recombinant TEV protease is more suitable for large-scale production and industrial use.

The invention discloses a method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos. The method comprises the following steps of: inserting coding sequences of TEV protease and enzyme cutting sites thereof between coding sequences of signal peptide and mature IL-37 proteins to form an IL-37-TEV fusion gene; according to the preference of tobacco codons, under the condition of guaranteeing no change of the amino acid sequence, optimizing the sequence of the fusion gene; connecting 5'-end upstream of the constructed IL-37-TEV fusion gene with a common promoter including other regulating areas, and connecting the 3'-end downstream with a transcription terminator; integrating a constructed recombinant expression vector onto cell chromosomes of the tobacco to culture a transgenic tobacco plant with high-efficiency stable expression of IL-37 and injecting the constructed recombinant expression vector to the tobacco leaves to instantaneously express target proteins. The method disclosed by the invention has the advantages that by construction of the plant expression vector, the mature proteins of IL-37 with high activity are expressed in the plants, and the obtained product has important application value for treatment of numerous inflammatory diseases and early detection and diagnosis of the inflammatory diseases.

The invention relates to a method for efficiently expressing hypersensitive protein by using a T4 phage display technology. The method comprises the following steps: (1) sequentially connecting an encoding gene of the hypersensitive protein with TEV protease and a T4 bacteriophage Soc protein gene segment to prepare a fusion recombinant protein encoding gene, wherein a encoding region of the hypersensitive protein is located at the upstream of a TEV-Soc fusion protein gene and having consistent expression cassettes; (2) transferring the coding gene of the fusion recombinant protein into a pUC18 plasmid; (3) transferring the plasmid into an escherichia coli host; (4) infecting the escherichia coli host by using Soc-T4 bacteriophage without Soc protein, and culturing the T4 bacteriophage ofwhich the host expresses and displays the fusion recombinant protein; (5) separating the T4 bacteriophage showing the fusion recombinant protein; and (6) separating the fusion recombinant protein fromthe T4 bacteriophage, and carrying out TEV protease digestion to obtain the hypersensitive protein.

The invention discloses a preparation method for beta-lactamase inhibitory polypeptide and an expression vector, a fusion protein and the like used therein. The method comprises the following steps: 1) constructing a recombinant expression vector used for expressing His tag fusion protein which is a fusion protein of a TEV protease cleavage site His tag of beta-lactamase inhibitory polypeptide; 2) transforming host cells so as to obtain a transformant; 3) culturing the transformant and expressing the His tag fusion protein; 4) carrying out purification by using metal chelated affinity chromatography so as to obtain the His tag fusion protein; 5) carrying out restriction enzyme on the His tag fusion protein with TEV protease; 6) carrying out separation and purification by using gel column chromatography so as to obtain beta-lactamase inhibitory polypeptide. According to the invention, the effect of restriction enzyme of TEV protease is ideal, and TEV protease used in the method has a low price, thereby enabling both purification difficulty and cost of beta-lactamase inhibitory polypeptide to be substantially reduced.

The invention discloses a spy capturer mutant, a preparation method thereof and application of the spy capturer mutant in a fluorescent protein system. Belongs to the technical field of biological coupling. According to the spyware catcher mutant, a flexible amino acid sequence is connected with the original N end and C end of a spyware SpyCatcher to obtain the spyware catcher mutant SpyCatcher-N, and a TEV protease recognizable sequence is inserted into the flexible amino acid sequence in the spyware catcher mutant SpyCatcher-N to obtain the spyware catcher mutant SpyCatcher-NTEV. The prepared spy capturer mutant has the advantages that on one hand, the high reactivity of a spy capturer is kept, and on the other hand, after a ligation reaction, Catcher protein can be digested by protease to reduce the molecular weight. Extra molecular weight is removed in a protease digestion mode.

The invention provides a preparation method of a high-purity cryptogein, which comprises the following steps: (1) sequentially connecting a coding gene of a target protein with TEV protease and T4 bacteriophage Hoc protein gene fragments to prepare a coding gene of the fusion recombinant protein; (2) transferring the coding gene of the fusion recombinant protein into a plasmid; (3) transferring the plasmid into an escherichia coli host; (4) infecting the escherichia coli host with Hoc-T4 bacteriophage deleted by Hoc protein, and culturing the host to express T4 bacteriophage displaying the fusion recombinant protein; (5) separating T4 bacteriophage displaying fusion recombinant protein; (6) separating the fusion recombinant protein from the T4 bacteriophage, and obtaining the target protein after TEV protease cleavage. The preparation method can simply and rapidly obtain high-purity cryptogein, and the obtained cryptogein solution can be directly used as an agricultural antibacterial bacteriostatic agent to achieve the effect of obviously promoting plant growth, thereby having good application prospect.