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A high-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzymes

A polypeptide coupling and protein technology, which is applied to the coupling or cyclization of proteins. The polypeptide-polypeptide coupling system based on disordered protein coupling enzymes can solve the problem of low coupling efficiency of ligases and achieve functional The effect of stability, high reaction efficiency and high purity

Active Publication Date: 2022-04-26
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two systems require the addition of an additional small molecule additive, trimethylamine N-oxide (TMAO) or glycerol, and their ligases are not very efficient

Method used

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  • A high-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzymes
  • A high-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzymes
  • A high-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Transformation and expression of fusion protein target gene

[0032] Plasmids of pQE-80 L ELP-SpyTag-ELP and pQE-80 L ELP-BDTag-ELP were constructed, and after being confirmed by sequencing, they were transferred into BL21 (DE3) competent cells and plated, and left overnight in the incubator for 37 o C culture. Pick out the monoclonal colony on the plate to the LB medium containing 0.10 mg / mL ampicillin sodium, in a shaking incubator for 37 o C was incubated overnight. The overnight culture medium was added to 1 L LB medium containing 0.10 mg / mL ampicillin sodium at a ratio of 1:100, at 37 o C shaking culture to OD 600 For 0.4-0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM to induce E. coli to express proteins. Shaking incubator temperature changed to 30 o C culture.

[0033] The plasmid of pACYCDuet-1 SpyTag-DHFR-BDTag (MCS1)-SpyStapler (MCS2) was constructed, and after being confirmed by sequencing, it was transfe...

Embodiment 2

[0034] Embodiment 2: the purification of fusion protein

[0035] After the expression was finished, the cells were collected by high-speed centrifugation at 4000 g for 20 minutes, and the supernatant was discarded. For the protein Spy Stapler enzyme (SpyStapler), spy stapler enzyme mutant (SpyStapler-EQ) and fusion protein SpyTag-DHFR-BDTag purified under natural conditions, use non-denaturing buffer A (20 mM sodium dihydrogen phosphate , 300 mM NaCl, 10 mM imidazole, pH = 8.0) resuspended. Resuspend at 4 o C Sonicate for 20 minutes in an ice-water bath (working for 5 seconds, interval of 5 seconds, intensity 40%). In; 4 o Under the condition of C, the crushed solution was centrifuged at 20,000 g for 20 minutes with a high-speed floor centrifuge, and the lysed supernatant was reserved. The supernatant was mixed with the equilibrated affinity resin Ni-NTA resin at 4 o C Rotate to mix well for 1 hour. Pour the mixed solution into the empty column, wait for the resin to set...

Embodiment 3

[0037] Example 3: Characterization of Fusion Proteins

[0038] 1 mL of the protein eluate was purified through a gel permeation column Superdex 200 Increase 10 / 300 GL on the AKTA protein purification system (AKTA Avant, GE Healthcare). The mobile phase was PBS, and the flow rate was 0.5 mL / min. By monitoring A 280 Collect peaks of interest.

[0039] The molecular weight of the purified product was determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) or high-performance liquid chromatography-electrospray mass spectrometry (LC-MS), and the instrument used was 5800 MALDI-TOF / TOF analyzer (ABSCIEX) . The result is as figure 1 As shown, in vivo SpyStapler-mediated peptide-peptide (BDTag-SpyTag) coupling reaction leads to in situ cyclization of SpyTag-DHFR-BDTag.

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Abstract

The invention discloses a high-efficiency polypeptide-polypeptide coupling system and method based on a disordered protein coupling enzyme. By designing and splitting the protein domain CnaB2 and optimizing it, a three-component system is obtained: spy tag (SpyTag), BD tag (BDTag) and spy stapler enzyme (SpyStapler), so that SpyStapler can catalyze the coupling between SpyTag and BDTag Linkage reaction, and prepare different fusion proteins containing SpyTag and BDTag two tags and carry out coupling or cyclization reaction. Based on the polypeptide-polypeptide coupling system of the present invention, an active protein with high purity and stable function can be obtained by means of gene coding; the spy stapler enzyme coupling spy label and BD label have high reaction efficiency, and some of the staples are stapled after the reaction The mechanical enzyme falls off, and the coupling part of the obtained cyclization product has a smaller molecular weight. The protein tag of the present invention is applicable to various functional proteins and can be fused and expressed at any position of the protein.

Description

technical field [0001] The invention relates to polypeptide coupling technology, in particular to a polypeptide-polypeptide coupling system based on a disordered protein coupling enzyme, and a method for coupling or realizing cyclization of proteins based on the system. Background technique [0002] Peptide / protein tags are widely used in protein purification, protein topology engineering, biological imaging and other fields. Protein tools such as SnoopLigase, Intein, Sortase, Butelase 1 and OaAEP1 are recognized for their high efficiency and specificity. It is considered to be a very effective biological peptide coupling method. [0003] The emergence of "molecular superglue" technology provides a new class of protein labeling strategies. The SpyTag and SpyCatcher systems, the SnoopTag and SnoopCatcher systems and their mutants have demonstrated the specific affinity labeling properties of "molecular glue". [0004] At present, there are few protein labeling tools based ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C12N15/70C07K19/00C12N9/06
CPCC12P21/00C12N15/70C12N15/62C07K14/78C12N9/003C12Y105/01003C07K2319/20C07K2319/21
Inventor 张文彬吴夏泠刘雅杰刘栋
Owner PEKING UNIV
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