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KR-20260065908-A - E. Expression vector for recombinant U-conotoxin TIIIA or TIIIALAMUT expression in E. coli

KR20260065908AKR 20260065908 AKR20260065908 AKR 20260065908AKR-20260065908-A

Abstract

The subject of the present invention is a construct for an expression vector for recombinant μ-conotoxin TIIIA or TIIIAlaMut expression, which comprises the nucleotide sequence of sequence no. 1 of μ-conotoxin TIIIA or the nucleotide sequence of sequence no. 4 of μ-conotoxin TIIIAlaMut, and both of which, at the 5' end, comprise a TRX:: TIIIA fusion protein containing a gene encoding a μ-conotoxin TIIIA gene and a leader protein, or a TRX::TIIIAlaMut fusion protein containing a gene encoding a μ-conotoxin TIIIAlaMut gene and a leader protein, and a sequence encoding six histidines (6His) linked by a serine-glycine-serine linker (SGS), wherein the leader protein is thioredoxin (TRX) modified by point mutagenesis in which the amino acid methionine at position 37 is replaced with lysine. Another subject of the present invention is an expression vector comprising a construct according to the present invention under the control of a homeostatic promoter. Another subject of the present invention is isolated E. coli cells comprising an expression vector according to the present invention. Another subject of the present invention is a method for producing μ-conotoxin TIIIA or TIIIAlaMut in E. coli using an expression vector comprising a construct according to the present invention, wherein the method is characterized by comprising the following steps: a) transforming E. coli cells with an expression vector comprising a construct according to the present invention encoding a TRX::TIIIA fusion protein having the amino acid sequence of SEQ ID NO. 2 or a TRX::TIIIAlaMut fusion protein having SEQ ID NO. 5 under the control of a homeostatic promoter; b) expressing the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; c) isolating and purifying the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; d) a step of forming disulfide crosslinks by applying the purified TRX::TIIIA or TRX::TIIIAlaMut fusion protein to glutathione treatment in GSH/GSSG and dialysis in buffer; e) a step of cleaving the TRX::TIIIA or TRX::TIIIAlaMut fusion protein having the formed disulfide crosslinks with cyanogen bromide; f) a step of purifying the cleaved TIIIA or TIIIAlaMut peptide.

Inventors

  • 마주르키에위츠-피사렉, 안나
  • 미키에위츠, 디아나
  • 마주르키에위츠, 알리나
  • 시아치, 토마즈
  • 아가타, 스테파넥
  • 매그달레나, 젠체우스카

Assignees

  • 사이언스4뷰티 에스피.지.오.오.

Dates

Publication Date
20260511
Application Date
20240904
Priority Date
20230905

Claims (15)

  1. As a construct for an expression vector for the expression of recombinant μ-conotoxin TIIIA or TIIIAlaMut, The above-mentioned construct comprises the nucleotide sequence of μ-conotoxin TIIIA sequence no. 1 or the nucleotide sequence of μ-conotoxin TIIIAlaMut sequence no. 4, and both comprise, at the 5' end, a sequence encoding six histids (6His) linked by a serine-glycine-serine linker (SGS) to a construct encoding a TRX::TIIIA fusion protein comprising a μ-conotoxin TIIIA gene and a gene encoding a leader protein, or a TRX::TIIIAlaMut fusion protein comprising a μ-conotoxin TIIIAlaMut gene and a leader protein. The above leader protein is a construct that is thioredoxin (TRX) modified by point mutagenesis in which the amino acid methionine at position 37 is replaced with lysine.
  2. An expression vector comprising a construct as defined in claim 1 under the control of a constant promoter.
  3. In paragraph 2, The above expression vector is a homeostatic promoter deoP1P2 .
  4. In paragraph 2 or 3, The above expression vector is an expression vector pDM, preferably a pDM having the nucleotide sequence of SEQ ID NO. 7, or a pDMR, preferably a pDMR having the nucleotide sequence of SEQ ID NO. 6.
  5. Isolated E. coli cells comprising an expression vector as defined in any one of paragraphs 2 to 4.
  6. In paragraph 5, The above cell is an isolated E. coli cell, which is an E. coli S4B cell.
  7. A method for producing μ-conotoxin TIIIA or TIIIAlaMut in E. coli using an expression vector comprising a construct as defined in claim 1, The above method comprises the following steps: a) transforming E. coli cells with an expression vector comprising a construct according to claim 1 encoding a TRX::TIIIA fusion protein having the amino acid sequence of SEQ ID NO. 2 or a TRX::TIIIAlaMut fusion protein having SEQ ID NO. 5 under the control of a homeostatic promoter; b) a step of expressing the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; c) a step of isolating and purifying the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; d) a step of forming disulfide crosslinks by applying the purified TRX::TIIIA or TRX::TIIIAlaMut fusion protein to glutathione treatment in GSH/GSSG and dialysis in buffer; e) a step of cleaving the TRX::TIIIA or TRX::TIIIAlaMut fusion protein having the disulfide crosslinked formed above with cyanogen bromide; f) A step of purifying the above-decomposed TIIIA or TIIIAlaMut peptide.
  8. In Paragraph 7, The method used in step a) is deoP1P2 , the constant promoter used.
  9. In Article 7 or Article 8, A method in which the expression vector used in step a) is pDM, preferably pDM having the nucleotide sequence of SEQ No. 7, or pDMR, preferably pDMR having the nucleotide sequence of SEQ No. 6.
  10. In any one of paragraphs 7 through 9, The above-mentioned transformed cells are E. coli S4B cells, method.
  11. In any one of paragraphs 7 through 10, A method in which, in step a), the transformation is performed by electroporation.
  12. In any one of paragraphs 7 through 11, In step b), the transformed expression a. The method wherein the culture of the E. coli strain is performed at 30°C.
  13. In any one of paragraphs 7 through 12, In step e), the cleavage is performed using cyanogen bromide provided in a molar excess of 100:1 for the methionine residue, in a method.
  14. In any one of paragraphs 7 through 13, In step e), the weight ratio of the cyanogen bromide to the cleaved fusion protein is 1.375:1, method.
  15. In any one of paragraphs 7 through 14, In step e), the cutting is performed in 0.1 M HCl at room temperature for 3 hours with stirring in a dark place, in a method.

Description

E. Expression vector for recombinant U-conotoxin TIIIA or TIIIALAMUT expression in E. coli The subject of the present invention is a composition for an expression vector for the expression of recombinant μ-conotoxin TIIIA or TIIIAlaMut, an expression vector comprising the same, isolated E. coli cells comprising the expression vector, and a method for producing μ-conotoxin TIIIA or TIIIAlaMut in E. coli using the expression vector comprising the composition. Conotoxins are substances with rapid paralyzing effects that, due to their properties, enable the regulation of specific heteromembrane receptors, ion channels, and transporters. In nature, conotoxins are found in the venom of sea snails belonging to the family Conidae and are used to immobilize and paralyze prey. These snails inhabit coral reefs and are mostly found in tropical and subtropical waters such as the South China Sea, the Australian coast, and the Pacific Ocean. There are approximately 700 species of cone snails, all of which are toxic. They are generally classified into three groups based on their feeding habits: worm predators, mollusk predators, and fish predators. Conotoxins are small peptides derived from cone snail venom and have evolved for the purpose of capturing prey and defending against predators. They are bioactive molecules belonging to a highly diverse class structurally and functionally, and are highly selective for various ligand-gated and voltage-gated ion channel subtypes. Conotoxins have proven to be a useful tool for studying the ligand binding domains of ion channels. Furthermore, they can be used as therapeutic agents that target specific ion channels. Ion channels are specialized cell membrane proteins that transport ions across the cell membrane. This is widely expressed in both excitable cells (neurons, muscles) and other cells (renal tubules, epithelial cells) (References [ Gao B., Peng C., et al. (2017). Cone snail: A Big Store of Conotoxins for Novel Drug Discovery. Toxins 2017, 9,397; 1-19. doi:10.3390/toxins9120397 ]; [ Sillar KT, Picton LD, Heilter WJ, (2016). The Neuroethology of Predation and Escape, First Edition, chapter 13. Neurotoxins for Attack and Defence. John Wiley & Sons, Ltd. Published. Oliviera BM, Cruz LJ (2001). Conotoxins, in retrospect. Toxicon 39; 7-14. doi:10.1016/S0041-0101(00)00157-4 ]). The mechanism of action of these peptides is to block specific neurotransmitters at neuronal synapses. Conversely, conotoxins themselves are generally microproteins with a length of less than 40 amino acids that enable heterogeneous recombination expression. The formation of highly precise (often multiple) disulfide bonds is particularly common in conotoxins and generally serves to enhance resistance to proteolysis as well as stabilize the major bioactive conformation critical to efficacy and selectivity (Reference [ Pennington MW, Czerwinski A., Norton RS (2017). Peptide therapeutics from venom: Current status and potential. Bioorganic & Medical Chemistry 2017; doi:org/10.1016/j.bmc.2017.09.029 ]). The venom of each individual cone snail contains up to 100 distinct peptides, each of which plays a specific role when injected into a target. It is the cumulative effect of these individual peptides that makes the venom lethal to prey. Conotoxins are classified into several classes based on their specificity. Omega-conotoxins inhibit neurotransmitter release by targeting and blocking voltage-sensitive Ca2 + channels. Alpha-conotoxins and psi-conotoxins induce ganglion and neuromuscular blockade by targeting and blocking nicotinic acetylcholine (ACh) receptors. Mu- and delta-conotoxins block voltage-sensitive Na + channels in muscles. Kappa-conotoxins block voltage-sensitive K + channels, which can also lead to increased neuronal excitability. Gamma-conotoxins target voltage-sensitive nonspecific cation channels, while sigma-conotoxins antagonize serotonin 5HT3 receptors (References [ Becker S., Terlau H. (2008). Toxins from cone snails: properties, applications and biotechnological production. Microbiol. Biotechnology 79; 1-9. doi:10.1007/s00253-008-1385-6 ]; [ Oliviera BM (1997). Conus venom peptides, receptor and ion channel targets and drug design: 50 million years of neuropharmacology. Mol Biol Cell 8; 2101-2109. doi:10.1091/mbc.8.11.2101 ]). Conotoxins are highly specific biological probes that provide scientists with tools to understand and distinguish closely related receptors. The simplicity of conotoxins has made them valuable for the advancement of neuroscience research and, consequently, the development of drugs and cosmetics. Many diseases, such as epilepsy, schizophrenia, Tourette syndrome, Parkinson's disease, and sclerosis, are associated with dysfunction of ligand-gated and voltage-gated channels. Conotoxins have proven to be highly promising as they are relatively small, potent, selective antagonists and agonists of specific cell membrane channel proteins, and thus can be used as excellent cosm