4 results about "Fusion Protein Expression" patented technology

This invention belongs to the field of plant gene editing technology, specifically relating to a bifunctional gene editing system and its application in single-parent maize. The invention provides a bifunctional gene editing system comprising: a fusion protein expression cassette and a guide RNA combined expression cassette; wherein the guide RNA combined expression cassette includes: pegRNA associated with the maize D9 gene; and sgRNA associated with the maize YUC4 and YUC2 genes. Transient expression validation using the bifunctional gene editing system in maize protoplasts revealed that the system achieves a precise editing efficiency of 3.5% at the G571V site of the D9 gene, a knockout efficiency of 40%-65% for the YUC4 gene, and a knockout efficiency of 37%-54% for the YUC2 gene. This demonstrates that the technical solution of this application can simultaneously and efficiently achieve precise point mutation and gene knockout within the same cell, thus enabling its application in the synergistic improvement of lodging resistance in maize.

This invention discloses a method for the bioenzymatic synthesis of ascorbate phosphate succinyl tripeptide-1, belonging to the field of bioengineering technology. This method involves constructing a fusion protein expression system through genetic engineering, releasing the target tripeptide GHK (glycylhistyllysine) through specific enzymatic cleavage, and then conjugating it to the succinyl group of ascorbate phosphate via an enzymatic catalytic esterification reaction, ultimately obtaining high-purity ascorbate phosphate succinyl tripeptide-1. Compared with traditional chemical synthesis methods, this method has advantages such as shorter synthesis steps, milder conditions, environmental friendliness, and high yield (>85%), making it suitable for large-scale industrial production and showing broad application prospects in the cosmetics and pharmaceutical fields.

The subject of this invention is a construct of an expression vector for expressing recombinant µ-conotoxin TIIIA or TIIIAlaMut, characterized in that it comprises the nucleotide sequence of µ-conotoxin TIIIA SEQ ID NO:1 or the nucleotide sequence of µ-conotoxin TIIIAlaMut SEQ ID NO:4, both sequences containing a sequence encoding six histidine residues (6His) at the 5' end, which is linked via a serine-glycine-serine linker (SGS) to a construct encoding a TRX::TIIIA fusion protein or a TRX::TIIIAlaMut fusion protein, wherein the TRX::TIIIA fusion protein comprises the µ-conotoxin TIIIA gene and a gene encoding a leader protein, and the TRX::TIIIAlaMut fusion protein comprises the µ-conotoxin TIIIAlaMut gene and a leader protein, wherein the leader protein is a thioredoxin (TRX) modified by site-directed mutagenesis, wherein the amino acid methionine at position 37 is replaced by lysine. Another subject of the invention is an expression vector comprising a construct according to the invention under the control of a constitutive promoter. Another subject of the invention is isolated *E. coli* cells comprising an expression vector according to the invention. Another subject of the invention is a method for producing µ-conotoxin TIIIA or TIIIAlaMut in *E. coli* using an expression vector comprising a construct according to the invention, characterized in that the method comprises the steps of: a) transforming *E. coli* cells with an expression vector comprising a construct according to the invention under the control of a constitutive promoter, said expression vector encoding a TRX::TIIIA fusion protein having the amino acid sequence SEQ ID NO:2 or having SEQ ID NO:2. a) TRX::TIIIAlaMut fusion protein NO:5; b) Expression of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; c) Isolation and purification of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; d) Formation of disulfide bonds by glutathione treatment of the purified TRX::TIIIA or TRX::TIIIAlaMut fusion protein in GSH / GSSG and dialyzing in buffer; e) Cleavage of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein with the formed disulfide bonds by cyanogen bromide; f) Purification of the cleaved TIIIA or TIIIAlaMut peptide.