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KR-20260065956-A - Catcher/Tag peptide linker with high ester bond formation efficiency

KR20260065956AKR 20260065956 AKR20260065956 AKR 20260065956AKR-20260065956-A

Abstract

The present invention relates to a Catcher/Tag peptide linker having high ester bond formation efficiency comprising a reverse catcher and a reverse tag, wherein the amino acid sequence of the reverse catcher is as shown in SEQ ID NO:1 and the amino acid sequence of the reverse tag is as shown in SEQ ID NO:2. The reverse tag and reverse catcher have significantly high covalent bonding efficiency, and the bonding reaches 100% at an extremely fast rate and can be completed within 170 seconds.

Inventors

  • 지앙, 링
  • 천, 야오
  • 스, 이
  • 리, 촹
  • 리우, 웨이
  • 주, 리잉

Assignees

  • 난징 테크 유니버시티

Dates

Publication Date
20260511
Application Date
20231020

Claims (10)

  1. A Catcher/Tag peptide linker having high ester bond formation efficiency, comprising a binding partner and a peptide tag, wherein the amino acid sequence of the binding partner is as shown in SEQ ID NO:1 and the amino acid sequence of the peptide tag is as shown in SEQ ID NO:2.
  2. A method for purifying a Catcher/Tag peptide linkage according to claim 1, (1) A step of obtaining a recombinant plasmid by cloning a peptide tag and a binding counterpart into a vector and introducing it into a host bacterium; (2) A step of inducing by adding IPTG after culturing a host bacterium having a recombinant plasmid to an OD600 of 0.6 to 0.8; (3) After induction is finished, centrifuge the bacterial suspension to collect cells, add PBS, and then perform ultrasonic disruption; (4) A method characterized by including the step of centrifuging the lysate to collect the supernatant, and obtaining purified protein through purification and dialysis.
  3. A method characterized in that, in paragraph 2, the vector described in step (1) is pET-22b.
  4. A method according to paragraph 2, characterized in that the enzymatic cleavage sites of the vector are Nde I and Xho I.
  5. A method according to claim 2, characterized in that the host bacterium in step (1) is Escherichia coli E. coli BL21(DE3).
  6. A method according to paragraph 2, characterized in that the purification described in step (4) is performed in a Ni-NTA resin.
  7. A method according to claim 2, characterized in that the protein purified in step (4) is dialyzed in a 4000 Da dialysis bag for 12 hours or more.
  8. The use of the Catcher/Tag peptide linkage according to claim 1 for protein separation.
  9. In paragraph 8, the above use is: (1-1) A step of obtaining a Cys-Reverse Catcher by connecting a cysteine (Cys) residue to the N-terminus of the bonding counterpart through a linker; (1-2) A step of obtaining a Reverse Tag-target protein by attaching the peptide tag to the N-terminus of the target protein; (1-3) A step of culturing the above-mentioned Cys-Reverse Catcher in a thiol group protein agarose coupling resin and coupling buffer solution, and then blocking treatment with Cys to obtain a resin to which the Cys-Reverse Catcher is bound; (1-4) A step of obtaining a bound resin by culturing the resin bound to the Reverse Tag-target protein and the Cys-Reverse Catcher in an ester binding reaction solution, and then removing the remaining ester binding reaction solution so that the ester binding reaction solution contains glycerol and CaCl2 ; (1-5) An application characterized by including the step of collecting the target protein after hydrolyzing the ester bonds by culturing the above-mentioned bonding resin in an ester bond hydrolysate.
  10. In claim 9, the use is characterized in that the amino acid sequence of the above-mentioned Linker is SEQ ID NO:3.

Description

Catcher/Tag peptide linker with high ester bond formation efficiency The present invention belongs to the field of molecular peptide design and, specifically, relates to a Catcher/Tag covalent linker with high ester bond formation efficiency. In 2014, Edward N. Baker's research team discovered and analyzed the structure of an Ig-like protein that forms an isopeptide bond between Thr and Gln, and its crystal structure PDB ID is 4ni6; based on 4ni6, the team separated it into Catcher and Tag in 2017. Although Thr11 of Catcher and Gln14 of Tag can form a covalent bond (ester bond) in a weakly acidic environment, glycerol and CaCl2 must be added to the system. Unlike typical molecular peptide pairs, the ester bond can be hydrolyzed if glycerol and Ca2 + are removed by dialysis after adjusting the environment pH to 8.0. Although this is applied in fields such as protein separation, the low efficiency of covalent bond formation limits the expansion of its applications. As a prior art achievement of the applicant, Chinese patent CN 116284278 A discloses a molecular peptide mutant EBCatcher having high ester bond formation efficiency and can provide binding efficiency with EBtag, although its binding efficiency is relatively high but the reaction rate is slow and does not meet the requirements of some application fields. To solve the problems of the aforementioned prior art, the present invention utilizes a rational design to modify a mutant based on EBCatcher/EBTag and forms a Reverse Catcher/Reverse Tag linkage, thereby enabling the rapid and high-efficiency formation of ester bonds. To achieve the above-mentioned objective, the present invention uses the following technical method: A peptide linker having high ester bond formation efficiency, comprising a reverse catcher and a reverse tag, wherein the amino acid sequence of the reverse catcher is as shown in SEQ ID NO:1 and the amino acid sequence of the reverse tag is as shown in SEQ ID NO:2. The amino acid sequence of the binding counterpart (Reverse Catcher) of SEQ ID NO:1 above is TIPEVKEGTLKTTVAADGVNGSSEKEALVSYENSKDGVDVKDTIDYKDLVPNEKYNLTGKLMHVKDDGSLEEVATKTTEVTAVENGSGQWELDFGNQKLQVGEKYVVFERAESVEDLIDTDNNYE. The amino acid sequence of the peptide tag (Reverse Tag) of SEQ ID NO:2 above is DTKQVVKHEDKNDKAQTLIVEKPNR. Another object of the present invention is to provide a method for purifying the peptide linkage, comprising the following steps: (1) A step of obtaining a recombinant plasmid by cloning Reverse Tag-GFP and Reverse Catcher-GFP into a vector and introducing it into a host bacterium; (2) A step of inducing by adding IPTG after culturing a host bacterium having a recombinant plasmid to an OD600 of 0.6 to 0.8; (3) After induction is finished, centrifuge the bacterial suspension to collect cells, add PBS, and then perform ultrasonic disruption; (4) A step of centrifuging the lysate to collect the supernatant, and obtaining purified protein through purification and dialysis. Preferably, the vector described in step (1) is pET-22b. Preferably, the enzymatic cleavage sites of the vector are Nde I and Xho I. Preferably, in step (1), the host bacterium is Escherichia coli E. coli BL21(DE3). Preferably, in step (2), the host bacteria having the recombinant plasmid are cultured in LB medium. Preferably, the induction conditions in step (2) are induced at 20°C for 12 to 14 hours, and the final IPTG concentration is 0.5 to 1 mM. Preferably, the centrifugation conditions in step (3) are centrifuged at 12,000 rpm for 3 to 5 minutes. Preferably, the ultrasonic crushing conditions described in step (3) are crushed at 300 to 400 W for 10 to 15 minutes. Preferably, the purification described in step (4) is performed in Ni-NTA resin. Preferably, the protein purified in step (4) is dialyzed in a 4,000 Da dialysis bag for more than 12 hours. Another objective of the present invention is to provide a use for the peptide linkage in protein separation. Preferably, the above use includes the following steps: (1-1) A step of obtaining a Cys-Reverse Catcher by connecting a cysteine (Cys) residue to the N-terminus of a Reverse Catcher through a Linker; (1-2) A step of obtaining a reverse tag-target protein by attaching the reverse tag to the N-terminus of the target protein; (1-3) A step of culturing the above-mentioned Cys-Reverse Catcher in a thiol group protein agarose coupling resin and coupling buffer solution, and then blocking treatment with Cys to obtain a resin to which the Cys-Reverse Catcher is bound; (1-4) A step of obtaining a bound resin by culturing the resin bound to the Reverse Tag-target protein and the Cys-Reverse Catcher in an ester binding reaction solution, and then removing the remaining ester binding reaction solution so that the ester binding reaction solution contains glycerol and CaCl2 ; (1-5) A step of collecting the target protein after hydrolyzing the ester bonds by culturing the above-mentioned bonding resin in an ester bond hydrolysate. Preferably,