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Method for preparing cysteine protease through cell-free protein synthesis system

A technology of cysteine ​​protease and cell-free protein, which is applied in the field of protein preparation to achieve the effect of high expression and simplified purification steps

Inactive Publication Date: 2017-06-06
WUHAN GENECREATE BIOLOGICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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The present invention has no report at home and abroad

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  • Method for preparing cysteine protease through cell-free protein synthesis system
  • Method for preparing cysteine protease through cell-free protein synthesis system
  • Method for preparing cysteine protease through cell-free protein synthesis system

Examples

Experimental program
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Effect test

Embodiment 1

[0030] 1. Synthesis and Cloning of Cysteine ​​Protease Gene

[0031] The cysteine ​​protease gene AsNODf32 of milk vetch was used to compare and analyze on the Japanese japonicum japonicus website to find homologous clones in japonicus japonicus; design specific primers to amplify cysteine ​​from japonicus japonicus genome Protease gene, its gene sequence is as shown in the nucleotide sequence of sequence table SEQ ID NO: 1, with synthetic DNA as PCR template, upstream primer is 5'-GATC GGATCC CTGCCGAAAGCGCCG-3', the downstream primer is 5'-ATG AAGCTT AATATCACGCACATAT-3', respectively introduce restriction sites for BamH Ⅰ and Hind Ⅲ. PCR reaction conditions: pre-denaturation at 95°C for 4min, denaturation at 94°C for 1min, annealing at 52°C for 1min, extension at 72°C for 2min, 30 cycles, and extension at 72°C for 6min.

[0032] 2. Construction of recombinant plasmids

[0033] The gene fragments amplified by PCR were recovered and digested with endonucleases BamH I and H...

Embodiment 2

[0039] Steps 1 to 4 are identical to Steps 1 to 4 of Example 1, wherein the precipitation process in step 5 is as follows:

[0040] The pellet was resuspended with PBS, centrifuged, and the supernatant was discarded, and this step was repeated twice. Resuspend the precipitate with 1 mL of 1.5% SKL solution, add DTT to a final concentration of 5 mM, shake at 30°C, 220r for 1 h until the precipitate is completely dissolved. Centrifuge and take the supernatant. Add 20% PEG4000 to a final concentration of 2%, 50 mM GSSG to a final concentration of 5 mM, and 100 mM GSSH to a final concentration of 8 mM. Place the centrifuge tube at 4°C for 12 hours. The protein after standing was taken out and dialyzed against TE buffer for 3 days. TE buffer was changed twice during dialysis. Centrifuge to obtain the supernatant. This is the final cysteine ​​protease solution. figure 2 Electropherogram of the cysteine ​​protease prepared for Example 2.

[0041] Wuhan Jinkairui Biological E...

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Abstract

The invention discloses a method for preparing cysteine protease through a cell-free protein synthesis system and belongs to the technical field of biology. The method comprises the following steps of (1) synthesizing and cloning a cysteine protease gene; (2) constructing recombinant plasmids pET28a-AsNODf32; (3) preparing a cell-free protein expression system and carrying out expression in the expression system to form a cysteine protease sediment; and (4) carrying out resuspended sedimentation treatment on the cysteine protease. The synthesized cysteine protease gene is expressed in the cell-free protein synthesis system, mRNA does not need to be prepared, the expression quantity is high and the subsequent purification step is simplified.

Description

technical field [0001] The invention relates to the field of protein preparation, in particular to a method for preparing cysteine ​​protease through a cell-free protein synthesis system. Background technique [0002] Cysteine ​​protease is the most important protease family, which is widely involved in various physiological processes of plants, such as seed germination, seedling development, stress response, plant tissue differentiation and senescence. Plant cysteine ​​proteases mainly belong to the papain-like cydteine ​​proteinases (C1A, papain-like cydteine ​​proteinases) and the bean protease family (C13the legumains), and the former has been studied in the most detail. Many papain enzymes contain signal peptides composed of 10-26 hydrophobic amino acids located in the endoplasmic reticulum. In the enzyme precursor, there is a domain consisting of 35-150 amino acids, which blocks the active site of the enzyme and is also critical for the correct folding and positioning...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N15/57C12N15/70
CPCC12N9/63C12N15/70C12N2800/101
Inventor 华权高沈鹤霄马峰罗绍祥肖颖舒芹徐春雷王静邓陈玲易汪雪李昆鹏
Owner WUHAN GENECREATE BIOLOGICAL ENG CO LTD
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