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Recombinant small ubiquitin-related modifier (SUMO) protease and preparation method as well as application thereof

A small molecule ubiquitin and modification technology, applied in the field of recombinant protease, can solve the problems of low stability, poor specificity, unable to maintain high activity, etc., to achieve the effect of enhanced stability, high specificity and convenient purification

Active Publication Date: 2011-11-09
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The existing proteases used to cleave SUMO from fusion proteins mainly have defects such as poor specificity, low stability, and inability to maintain high activity in a wide range of reaction environment systems, which need to be improved

Method used

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  • Recombinant small ubiquitin-related modifier (SUMO) protease and preparation method as well as application thereof
  • Recombinant small ubiquitin-related modifier (SUMO) protease and preparation method as well as application thereof
  • Recombinant small ubiquitin-related modifier (SUMO) protease and preparation method as well as application thereof

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Experimental program
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Embodiment 1

[0026] Embodiment 1 Preparation and purification of recombinant SUMO protease (Ulp1p-GST) of the present invention

[0027] 1. Construction of recombinant expression plasmids

[0028] The ULP1P nucleotide sequence and the poly-His tag nucleotide sequence were joined together and digested with BamH I and Xho I, and then subcloned into the plasmid pGEX-6p-1 to obtain the recombinant expression plasmid pGEX-6p-1 -His; its construction process see figure 1 .

[0029] 2. Conversion

[0030] Thaw rosetta competent cells on ice, add 1 μl of recombinant expression plasmid, place on ice for 30 minutes, heat shock at 42°C for 50 seconds, add 200-300 μl of liquid LB (without AMP), shake at 37°C for 1 hour, spread on the plate, and overnight at 37°C to cultivate.

[0031] 3. Express

[0032] (1) Activation: Pick a single colony and put it in 10-20ml LB, shake overnight at 37°C.

[0033] (2) Secondary activation: 1 / 100 inoculation (500ml to 5ml), shake at 37°C for 2-3 hours to OD 60...

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Abstract

The invention discloses a recombinant small ubiquitin-related modifier (SUMO) protease and a preparation method as well as application thereof. The recombinant protease is composed of a GST (glutathione-s-transferase) tag, a ULP1P (ubiquitin-like-specific protease 1) sequence and a poly-His (histidine) tag, wherein the amino acid sequence of the recombinant protease is shown in SEQ ID NO:2. The recombinant SUMO protease disclosed by the invention recognizes a complete SUMO tag protein sequence and can efficiently cut off the SUMO from fusion protein. Compared with recognition sites of proteases such as EK, TEV and the like, the restriction reaction has very high specificity and keeps higher activity in reaction environment systems in a wider range. The recombinant SUMO protease disclosed by the invention is not only provided with the poly-His tag to be convenient for affinitive layer purification after cutting of the fusion protein, but also provided with the GST tag to be convenient for purifying the protease and enhancing the stability of the protease.

Description

technical field [0001] The invention relates to a recombinant protease, in particular to a recombinant small molecule ubiquitin-like modifier protease and a preparation method thereof. The invention also relates to the application of the recombinant protease as a tool enzyme for cleavage and purification of recombinant proteins, belonging to the field of recombinant proteases. Background technique [0002] The small ubiquitin-related modifier (SUMO) protease is a member of the cysteine ​​protease superfamily, and it mainly performs two physiological functions in eukaryotes: (1) cutting the SUMO precursor C - a few amino acid residues at the end to expose the C-terminal Gly-Gly residues to make it a mature SUMO with functions such as modification and transport; (2) hydrolyzing SUMO and the target protein conjugate into SUMO and target protein, This process is called de-SUMOization. Research data show that various SUMO proteases contain a conserved C-terminal ULP domain of ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N15/57C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P21/06
Inventor 李德山傅俊华任桂萍王文飞刘铭瑶
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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