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Cleavage efficiency enhanced substrate mutant of HRV (Human Rhinovirus) 3C protease and application thereof

A protease substrate, variant technology, applied in the field of bioengineering, can solve the problems of difficult screening, large workload, low efficiency, etc., and achieve the effect of efficient cutting advantages

Active Publication Date: 2018-01-19
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has a large workload, complicated operation, low efficiency, and large operational errors, which is not conducive to the efficient development of experiments, so it is difficult to screen for HRV 3C protease substrate sequence mutants with enhanced cleavage efficiency

Method used

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  • Cleavage efficiency enhanced substrate mutant of HRV (Human Rhinovirus) 3C protease and application thereof
  • Cleavage efficiency enhanced substrate mutant of HRV (Human Rhinovirus) 3C protease and application thereof
  • Cleavage efficiency enhanced substrate mutant of HRV (Human Rhinovirus) 3C protease and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Construction of HRV 3C protease and its substrate mutants inducible expression plasmids in Saccharomyces cerevisiae

[0023] (1) The substrate mutant gene fragment was amplified by three rounds of polymerase chain reaction (PCR). Among them, site-directed mutagenesis was carried out in the first round of PCR amplification to obtain the mutant substrate genes of HRV 3C protease substrate cleavage sites P1, P1', P2'; the second round of PCR amplification was used to determine whether the gene fragment had a retained signal peptide gene ; In the third round of PCR amplification, the restriction endonuclease cuts the gene sequence to obtain the complete gene. Among them, there are 20 kinds of amino acid saturation mutants with stranded signal peptide genes at the P1 site, and 3 kinds of mutant genes with amino acids Q, N, and F without the stranded signal peptide genes; There are 20 kinds of amino acid saturation mutants with stranded signal peptide genes, and 3 ...

Embodiment 2

[0031] Example 2 Induced expression of plasmids containing HRV 3C protease and substrate mutants thereof in Saccharomyces cerevisiae

[0032] Transform the final vector into Saccharomyces cerevisiae EBY100, and the reagents used for transformation are: EBY100 competent cells, 20 μL; recombinant plasmid, 1.5 μL; Single strand carrier DNA, 25 μL; 1M LiAC, 36 μL; PEG4000 (50% W / V), 240 μL After mixing the system, put it in a water bath at 30°C for 30 minutes, transfer it to 42°C for heat shock for 20 minutes, collect the bacteria by centrifugation, add 1mLYPD liquid culture and culture on a shaker at 30°C for 1.5hr, wash the cells once with ultrapure water, and then spread them on YNB- CAA-Glucose solid medium, cultivated in a 30°C incubator for 2-3 days. The composition of YNB-CAA-Glucose solid medium is 20g / L glucose, 6.7g / L YNB, 5.4g / L Na 2 HPO 4 ,8.6g / L NaH 2 PO 4 ·H 2 O and 5g / L casamino acids, pH7.4, 15g / L agar.

[0033] Inoculate a single colony in the liquid medium ...

Embodiment 3

[0034] Example 3 Application of flow cytometry to detect the reaction results of HRV 3C protease and its substrate mutants

[0035] take 10 6 Add 200 μL of Buffer A to mix well, then centrifuge to pellet the cells, 3000 rpm, 4°C, 1 min. Then wash the cells with Buffer B solution, remove the supernatant, then add 20 μL Buffer B solution, 0.15 μL FITC, 0.15 μLAPC to each sample, label in the dark at 4°C for 15 minutes, and transfer to room temperature for 30 minutes in the dark. Centrifuge to remove the supernatant, then wash once with 150μL Buffer B, continue to wash once with 1x PBS, and resuspend with 300μL. The resuspended cells were analyzed by CytoFLEX flow cytometer, and the fluorescent signal channels detected were FITC (525 / 40nm) and APC (660 / 20nm). The cleavage efficiency of HRV 3C protease to its substrate mutants was obtained from the analysis of the results given in the flow cytometer (see image 3 ).

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Abstract

The invention utilizes a yeast endoplasmic reticulum sequestration screening system (YESS) to quickly analyze the specificity of a cleavage site of a substrate of protease, and finally provides a cleavage efficiency enhanced substrate sequence mutant of HRV (Human Rhinovirus) 3C protease and application thereof. The substrate mutant includes an amino acid sequence as shown in SEQ ID NO.1 in a sequence table, and has a better practical application advantage in the removal of a purification tag in a protein purification process, and therefore, the further application and research of the HRV 3C protease are facilitated.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a HRV 3C protease substrate sequence mutant with enhanced cleavage efficiency and its application. Background technique [0002] Human rhinovirus (HRV) is a single-stranded positive-sense small RNA virus. The 3C protease encoded by it belongs to the cysteine ​​protease family, which can recognize the polypeptide sequence LEVLFQ↓GP and cut between Gln and Gly. HRV 3C protease has the characteristics of strong specificity and high enzyme activity, and it still has high enzyme activity even at a low temperature of 4°C. Currently, HRV 3C protease is widely commercialized, especially for the removal of purification tags during protein purification. However, as a high-value commercial enzyme, the substrate specificity of HRV3C protease has not been reported. [0003] In addition, the study of protease substrate cleavage site specificity is a necessary process for c...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C07K1/14C12R1/19
Inventor 易犁范贤彭文舫周瑜喻婵张桂敏
Owner HUBEI UNIV
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