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 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 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 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.