36 results about "TEV protease" patented technology

The invention discloses a recombinant I-type humanized collagen polypeptide as well as a preparation method and application thereof. The recombinant I-type humanized collagen polypeptide provided by the invention comprises n repeats of a sequence shown in SEQ ID No. 1, wherein n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeat sequences are directly connected; and optionally, the N tail end of the recombinant I-type humanized collagen polypeptide comprises an amino acid sequence which can be excised by TEV protease. The recombinant I-type humanized collagen polypeptide provided by the invention has the activity of promoting cell adhesion, the amino acid sequence of the recombinant protein is selected from a natural collagen amino acid sequence, and the recombinant protein does not generate an immune response when being applied to a human body; and moreover, the preparation method is simple, and high-yield collagen can be obtained at low cost.

A cis-TEVP fusion protein including a TEVP protease, a TEVP cleavage site and a target protein provide a platform for expression of the target protein. A trans-TEVP fusion protein including a TEVP cleavage site and a target protein, the amino-terminal portion of the target protein adjacent to the C-terminal portion of the TEVP cleavage site, the amino acidic residue in position P2 of the TEVP cleavage site being a Valine also produces the target protein by the same process. A cis-TEVP fusion protein system comprising the first fusion protein and a suitable host cell; a trans-TEVP fusion protein system comprising the second fusion protein and a suitable host cell; associated methods to produce target proteins, and kits of parts are also disclosed herein.

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.

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 genetic engineering, and particularly relates to an expression method of AEP cyclase in pichia pastoris and application thereof. The AEP cyclase is expressed in pichia pastoris, the gene tandem copy number of the AEP cyclase reaches up to 5 copies by a gene tandem way, and the expression amount of the AEP cyclase is highest up to 0.89+ / -0.03 mg / mL when the gene copy number reaches 3. The invention verifies the cyclization capacity of the AEP cyclase in a novel mode, constructs a tandem substrate, selects a protein as the substrate which can be better observed in SDS-PAGE and has the cyclization possibility, constructs a tandem substrate containing the substrate sequence of the TEV protease; and identifies whether the AEP cyclase is cyclized or not according to the size change of the substrate after the protease is cut or the number of bands. The method is simple and convenient to operate and strong in practicability.

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 provides a TEV protease variant, a fusion protein, a preparation method and uses thereof, particularly a screened TEV protease variant with unique properties, and a fusion protein. According to the invention, polypeptides can be rapidly and efficiently prepared through TEV protease variant and polypeptide fusion expression so as to solve the problems existing in the current polypeptide recombinant production process.

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 Hoc protein gene segment to prepare a fusion recombinant protein encoding gene, wherein the encoding region of the hypersensitive protein is located at the upstream of a TEV-Hoc fusion protein gene and having consistent expression cassettes; (2) transferring a 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 Hoc-T4 bacteriophage deleted by Hoc protein, and culturing the host to express and display the T4 bacteriophage of the fusion recombinant protein; (5) separating the T4 bacteriophage showing the fusion recombinant protein; and (6) separating the fusion recombinant protein from the T4 bacteriophage, and carrying out TEV protease digestion to obtain the hypersensitive protein.

The invention discloses a method for high-throughput screening of single-domain antibodies using isPLA. The steps are as follows: constructing an sdAb library containing 21 random amino acid sequences in the CDR3 region, and fusing a 3×Flag tag to the C-terminus of the sequence; co-transfection Cells, fixed cells for isPLA; followed by sorting, amplification, and recombination, and after multiple rounds of screening and sequencing, constructs that sequentially contain glutathione S-transferase GST, tobacco mosaic virus TEV protease cleavage site, and recognize SQSTM1. The sdAb sequence, the translocation domain of Pseudomonas exotoxin ETA and the 3×Flag-tagged recombinant protein were expressed in E. coli, followed by purification and enzyme digestion. The method can screen sdAbs that specifically recognize different antigenic determinants from the constructed high-capacity sdAb library, and has low cost and high efficiency. No preparation of animal samples or hybridomas is involved.

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 preparing high-purity cryptoprotein. The method comprises the steps of (1) sequentially connecting a coding gene of a target protein with a TEV protease and a T4 phage Soc protein gene fragment to prepare a fusion recombinant protein coding gene; (2) transferring the gene encoding the fusion recombinant protein into a plasmid; (3) transferring the plasmid into abacterium coli host; (4) infecting the bacterium coli host with a Soc-deficient Soc-T4 phage, and cultivating the host to express the T4 phage displaying the fusion recombinant protein; (5) isolatingthe T4 phage displaying the fusion recombinant protein; (6) isolating the fusion recombinant protein from the T4 phage, and obtaining the target protein by TEV protease digestion. The method can obtain the high-purity cryptoprotein easily and quickly, an obtained cryptoprotein solution can be directly used as an agricultural antibacterial bacteriostatic agent to achieve the effect of promoting plant growth, and the method has a good application prospect.

The invention belongs to the technical field of genetic engineering, and in particular relates to an expression method and application of AEP cyclase in Pichia pastoris. In the present invention, by expressing in Pichia pastoris, the number of copies of the AEP cyclase gene in series can reach up to 5 copies through the method of gene tandem, and when the gene copy number reaches 3, the expression of AEP cyclase is the highest, reaching 0.89± 0.03mg / mL. The present invention adopts a new method to verify the cyclization ability of AEP cyclase. By constructing a tandem substrate, a protein that can be better observed in SDS-PAGE and has the possibility of cyclization is selected as the substrate , and construct a tandem substrate containing the substrate sequence of TEV protease, and identify whether it is cyclized according to the change in the size of the substrate after protease cleavage or the number of bands, the operation is simple, convenient, and practical.

The invention relates to the field of biotechnology, and in particular to an expression vector, expression system and application of a recombinant TEV protease. The recombinant TEV protease expressionvector is obtained by inserting a His-TEVp recombinant protein into a pET-21a vector. The recombinant TEV protease expression system comprises introducing the recombinant TEV protease expression vector into E. coli to obtain a recombinant TEV protease expression strain. The His-TEVp recombinant protein is generated by inserting a four-point mutant TEVp gene into the pET-21a plasmid to obtain a recombinant plasmid pET-21a-TEVp. The TEV protease expression system is utilized to express the TEV protease, and then a large amount of high-purity TEV protease can be obtained by extraction and purification; the purity of the TEV protease can reach more than 90%; and the concentration of TEVp in the production bacterial liquid can reach 1.42 mg / ml, and high-purity TEV protease of more than 40 mg can be obtained in each liter of bacterial liquid. The expression vector and expression system of the recombinant TEV protease provided by the present invention lay a foundation for the popularizationand application of the TEV protease.

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.

The invention discloses a recombinant type I humanized collagen polypeptide and its preparation method and application. The recombinant type I humanized collagen polypeptide provided by the present invention comprises n repeats of the sequence shown in SEQ ID No.1, n is an integer greater than or equal to 1, wherein when n is an integer greater than or equal to 2, each repeat The sequences are directly connected; optionally, the N-terminal of the recombinant type I humanized collagen polypeptide contains an amino acid sequence that can be excised by TEV protease. The recombinant type I humanized collagen polypeptide provided by the present invention has the activity of promoting cell adhesion, the amino acid sequence of the recombinant protein is selected from the amino acid sequence of natural collagen, it will not produce an immune response when applied to the human body, and its preparation method is simple, Higher yields of collagen can be obtained at low cost.