Synthesis system, preparation, kit and preparation method of in-vitro DNA-to-Protein (D2P)

A synthesis system and system technology, applied in the direction of recombinant DNA technology, introduction of foreign genetic material using vectors, fermentation, etc., can solve the limitation of biosynthesis efficiency and yield, complex experimental requirements, increase operation time, manufacturing cost and experimental complexity, etc. question

Active Publication Date: 2018-10-12
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both of these systems are relatively complex and have high experimental requirements. Users need to perform in vitro transcription experiments in advance to obtain enough mRNA templates, or provide a large amount of DNA or high-concentration plasmids as templates in advance (the quality of the template is even greater than that of synthetic protein quality)
These disadvantages increase the user's operation time, manufacturing cost, and experimental complexity, and also greatly limit the efficiency and yield of biosynthesis

Method used

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  • Synthesis system, preparation, kit and preparation method of in-vitro DNA-to-Protein (D2P)
  • Synthesis system, preparation, kit and preparation method of in-vitro DNA-to-Protein (D2P)
  • Synthesis system, preparation, kit and preparation method of in-vitro DNA-to-Protein (D2P)

Examples

Experimental program
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preparation example Construction

[0248] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0249] (i) providing yeast cells;

[0250] (ii) washing the yeast cells to obtain washed yeast cells;

[0251] (iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0252] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0253] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0254] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0255] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000 g, preferably 8000-30000 g.

[0256] In the present invention, the centrifugation time is not particu...

Embodiment 1

[0287] Example 1: Amplification of plasmid templates using phi29 DNA polymerase

[0288] 1.1 Preparation of DNA amplification system: random primers at a final concentration of 20-30 μM, plasmid template at 0.05-0.15 μg / mL, dNTP at 0.5-1 mM, 2 × BSA, and phi29 DNA aggregation at 0.05-0.1 mg / mL Enzyme, 1 × phi29 reaction buffer (consisting of 50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH7.5).

[0289] 1.2 Amplification reaction of plasmid template DNA in vitro: Place the above-mentioned reaction system in an environment of 20-30 °C for 6-12 h, usually overnight.

Embodiment 2

[0290] Example 2: In vitro protein synthesis using amplified template DNA

[0291] 2.1 In vitro protein synthesis system: final concentration of 22 mM 4-hydroxyethylpiperazineethanesulfonic acid at pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture (adenoside Purine nucleoside triphosphate, guanosine triphosphate, cytidine nucleoside triphosphate and uridine nucleoside triphosphate), 0.08-0.24 mM amino acid mixture (glycine, alanine, valine, leucine, Isoleucine, Phenylalanine, Proline, Tryptophan, Serine, Tyrosine, Cysteine, Methionine, Asparagine, Glutamine, Threonine, Aspartic Acid, Glutamine acid, lysine, arginine, and histidine), 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg / mL creatine phosphokinase, 0.027-0.054 mg / mL T7 RNA polymerase, 1% - 4% polyethylene glycol, 0.5% - 2% sucrose, and finally 50% by volume of yeast cell extract;

[0292] 2.2 In vitro protein synthesis reaction: Add 0.5-3 μL of amplif...

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Abstract

The invention provides a theoretical design and technical design of cell-free protein synthesis for DNA replication, transcription and translation coupling, a preparation, a kit and a preparation method. Specifically, with the application of the in-vitro cell-free synthesis system provided by the invention, complex protein can be synthesized, and moreover, DNA and mRNA can be synthesized; and effective, high-throughput and quite convenient protein synthesis can be completed with the use of a DNA template by a minute quantity (nanogram-microgram).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an in vitro DNA-to-Protein (D2P) synthesis system, preparation, kit and preparation method. Background technique [0002] The traditional biosynthesis system refers to a molecular biology technique that expresses foreign genes through model organisms such as bacteria, fungi, plant cells or animal cells. With the development of science and technology, cell-free expression system, also known as in vitro protein synthesis system, came into being. The in vitro protein synthesis system refers to the synthesis of the target protein by using the exogenous target mRNA or DNA as a template, and artificially controlling the addition of substrates required for protein synthesis, transcription and translation-related protein factors and other substances. The in vitro protein synthesis system expresses proteins without the steps of plasmid construction, transformation, cell culture, cell collect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/85C12P21/00
CPCC12N15/81C12N15/85C12P21/00
Inventor 郭敏章小铃王海鹏王静徐开陈秋锦于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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