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A kind of preparation method of restriction endonuclease fsei

A technology of restriction endonuclease and encoding gene, which is applied in the field of preparation of restriction endonuclease FseI, can solve the problems of cumbersome separation and purification procedures, high protein price, low protein yield, etc. Market prospects, the effect of improving expression levels

Active Publication Date: 2022-05-24
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low expression level, low protein yield, and cumbersome separation and purification procedures of commercialized FseI, the price of the protein is relatively high, and few biological companies produce this kind of restriction enzyme.

Method used

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  • A kind of preparation method of restriction endonuclease fsei
  • A kind of preparation method of restriction endonuclease fsei
  • A kind of preparation method of restriction endonuclease fsei

Examples

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Effect test

Embodiment 1

[0065] Example 1 Construction of pQE-80L-FseI prokaryotic expression plasmid

[0066] The codon-optimized FseⅠ gene was cloned into the pQE-80L vector containing 6×His (histidine) tag to construct the pQE-80L-FseⅠ prokaryotic expression plasmid. The specific method is as follows:

[0067] 1. Synthesis of FseⅠ gene

[0068] In order to realize the high-efficiency expression of FseI, the present invention firstly performs codon optimization on the coding gene of FseI according to the codon preference of Escherichia coli. In the process of codon optimization, the present invention found that the expression level and purity of FseI protein obtained by using conventional codon optimization software or simply following the codon preference of the E. coli expression system for codon optimization of FseI cannot satisfy the The enzyme was prepared in large quantities, and there was no obvious rule between the codon optimization position and optimization degree and the expression level...

Embodiment 2

[0091] Example 2 Expression of FseI restriction endonuclease

[0092] 1. Preparation of Escherichia coli Origami Competent Cells

[0093] (1) Pick the pre-stored Origami strain and streak it on empty LB solid medium, and cultivate overnight at 37°C;

[0094] (2) Pick a single clone colony in 25mL of empty medium, cultivate at 37°C for more than 12h, to OD 600 >1.5;

[0095] (3) Take 3mL of bacterial solution in 300mL empty LB medium (1:100) in the ultra-clean bench, shake it to OD at 18°C ​​(200rpm) 600 is about 0.55;

[0096] (4) Bacterial liquid in ice bath for 10min, while pre-cooling Inoue transformation buffer;

[0097] (5) 4 °C, 4000rpm centrifugation for 10min to collect bacteria, discard the supernatant, invert on absorbent paper for 2min, blot dry the residual liquid, and resuspend the bacteria with 80mL of Inoue transformation buffer;

[0098] (6) 4 °C, 4000rpm centrifugation for 10min to collect bacteria, discard the supernatant, invert on absorbent paper for 2...

Embodiment 3

[0120]Example 3 Purification of FseI restriction endonuclease

[0121] 1. Protein purification by nickel column

[0122] (1) Use a 0.22 μm filter to filter the supernatant;

[0123] (2) Incubate the supernatant with Beads at 4°C for 1-2 hours, at which time FseI protein binds to Beads;

[0124] (3) adding the supernatant into the purification column, allowing the liquid to flow out naturally under the action of gravity, and the target protein bound to the Beads remains in the purification column, and the purification column is always kept in the chromatography cabinet to maintain a low temperature environment;

[0125] (4) Wash the purification column with Wash Buffer (20mM Tris-HCl, pH8.2; 25mM imidazole; 150mM NaCl) for 5-10 column volumes to remove the impurity proteins that are not bound to Beads, and use fast staining reagents to detect proteins in real time until When the quick-staining reagent no longer turns blue, it can be judged that no impurity protein has been el...

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Abstract

The invention relates to the field of biotechnology, in particular to a method for preparing a restriction endonuclease FseI. The present invention provides a recombinant expression vector, which contains one or more copies of the encoding gene of the restriction endonuclease FseI, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1. The present invention also provides a recombinant Escherichia coli containing the recombinant expression vector and a method for preparing the restriction endonuclease FseI by using the recombinant Escherichia coli. The preparation method of the invention can prepare high-purity and high-activity FseI restriction endonuclease in a relatively short time, greatly reduces the preparation cost of FseI, and improves the preparation efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a restriction endonuclease FseI. Background technique [0002] Restriction endonucleases are a class of enzymes that can recognize a specific deoxynucleotide sequence and cut the phosphodiester bond between two deoxyribonucleotides in a specific part of each chain, abbreviated as restriction enzyme, The position of its cleavage can be inside the recognition site or outside the recognition site. [0003] In 1970, Smith et al. found that bacteria have enzymes that can degrade phage DNA invading their bodies. This enzyme is named Hind II and has specific recognition and cleavage sites. Researchers found that the cut DNA fragments can be connected again, and began to realize that this restriction endonuclease is an important tool for studying biology, which will revolutionize the field of biotechnology. Restriction endonucleases are widely found in prokaryotes. I...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/70C12N1/21C12R1/19
CPCC12N9/16C12N15/70C12N2800/22
Inventor 董娜文伟彰
Owner CHINA AGRI UNIV
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