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Restriction endonuclease DpnI preparation and preparation method thereof

A technology of restriction endonucleases and preparations, applied in the field of proteases and restriction endonucleases, which can solve the problems of decreased protein activity, loss of protein yield, etc.

Inactive Publication Date: 2021-03-23
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After the existing commercialized DpnI is expressed by protein, it needs to go through separation and purification steps, and then re-dissolved in the storage solution, and stored at a low temperature of -20°C, and the separation and purification steps will inevitably lead to the loss of protein yield. , may also lead to decreased protein activity

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  • Restriction endonuclease DpnI preparation and preparation method thereof
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  • Restriction endonuclease DpnI preparation and preparation method thereof

Examples

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preparation example Construction

[0154] The invention provides a restriction endonuclease DpnI preparation and a preparation method thereof; the restriction endonuclease DpnI preparation is prepared by an in vitro cell-free protein synthesis system, and after in vitro protein synthesis, the target protein in the solution contains a DpnI structure, The collected supernatant with DpnI enzyme activity can be used to obtain the restriction endonuclease DpnI preparation, which can be packaged or prepared into a storage solution for packaging; it can also be prepared as a storage solution that can be stored at low temperature. The preparation method is simple, avoids the loss of protein content caused by traditional separation and purification steps, and has high enzyme cutting activity, and can also avoid the possible loss of activity caused by traditional purification steps, and can be widely used in molecular biology, bioengineering, food, Agriculture, feed, daily necessities, washing, environment and other field...

Embodiment 6

[0212] In Example 6, in the gene expression system encoding DpnI, insoluble substances will precipitate out of the system as the reaction progresses. In the co-expression system of DpnI, in Examples 1-3 encoding eGFP-2A-DpnI, eGFP-DpnI, eGFP-TEV-DpnI, encoding eGFP-2A-DpnI, mScarlet-2A-DpnI, eYFP-2A- In Examples 10-14 of DpnI, MBP-2A-DpnI, and DsbA-2A-DpnI, the precipitation of the system was significantly reduced. refer to Figure 4 (Example 1-3), the supernatant rate can reach 70%. The supernatant ratio refers to the content ratio of the target protein expressed in the system in the supernatant after solid-liquid separation. The supernatant rate can be obtained by using the fluorescent protein solubilization label SeFP (such as eGFP) as an indicator. Specifically, taking eGFP as an example: the RFU value of the reaction liquid after the reaction in the in vitro cell-free synthesis system is used as the total value of eGFP. Measure the RFU value of the supernatant obtained...

Embodiment 1-6

[0388] Construct the expression vector encoding DpnI protein and its eGFP fusion protein:

[0389] The artificially constructed plasmid vector designed for Kluyveromyces lactis cell extract contains T7 promoter, LAC4 terminator, 5' and 3' UTR and other functional elements. The plasmid vector can express various proteins in vitro in the Kluyveromyces lactis cell extract, including but not limited to DpnI, eGFP and the fusion protein of the two. It should be noted that the plasmid expression vector in this example is only used to specifically illustrate the embodiment of the present invention, and does not limit the scope of the present invention, as long as DpnI or its fusion protein can be expressed in a cell-free in vitro protein synthesis system (especially It is the fusion protein of DpnI and solubilization tag, the present embodiment is the eGFP fusion protein of DpnI), can be used as the plasmid carrier of the present invention; And not limited to the Kluyveromyces cell e...

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Abstract

The invention provides a restriction endonuclease DpnI preparation and a preparation method thereof. The restriction endonuclease preparation is prepared through an in-vitro protein synthesis reaction, specifically, an in-vitro acellular protein synthesis system is constructed, a DNA template containing a coding gene of DpnI is added, the in-vitro protein synthesis reaction is carried out, a target protein with a DpnI structure is synthesized, solid-liquid separation is carried out, supernatant is collected, the enzyme activity is measured, a supernatant solution with restriction endonucleaseactivity is selected, the restriction endonuclease DpnI preparation is obtained, and the preparation is packaged or is prepared into a storage solution and then packaged. According to the product, a preparation method is simple, the protein content loss caused by traditional separation and purification steps are avoided, the enzyme digestion activity is high, and the preparation can be widely applied to the fields of molecular biology, bioengineering, food and the like.

Description

technical field [0001] The invention relates to the technical field of proteases, in particular to the technical field of restriction endonucleases, in particular to a restriction endonuclease DpnI preparation and a preparation method thereof. Background technique [0002] DpnI, is a restriction endonuclease that can specifically and efficiently recognize adenine methylated G in DNA m ATC sequence (5'-G m6 ATC-3'), and specifically cuts after the methylated adenine site, but cannot cut off the non-methylated GATC sequence, and is mainly used for degrading methylated plasmid templates, gene site-directed mutations in vitro, etc. Application fields include but not limited to biomedicine, molecular biology, bioengineering, food, agriculture, feed, daily necessities, washing, environment and other fields. References "Weiguo Han, Miao Shi and Simon D Spivack. Site-specific methylated reporter constructs for functional analysis of DNA methylation [J]. Epigenetics, 8(11): 1176-11...

Claims

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Application Information

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IPC IPC(8): C12N9/22C07K19/00
CPCC07K2319/60C12N9/22
Inventor 郭敏徐丽琼于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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