Cell-free freeze-drying preparation for in-vitro protein synthesis, and preparation method and application of cell-free freeze-drying preparation

A technology for protein synthesis and freeze-dried preparations, applied in the biological field, can solve the problems of reduced protein synthesis capacity, inconvenient operation, affecting protein activity, etc.

Active Publication Date: 2019-12-10
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] One disadvantage of the cell-free expression system is the preservation of cell extracts. The liquid must be stored at -80°C and cannot be frozen and thawed repeatedly, otherwise it will seriously affect the ability of cell extracts to synthesize proteins. Keep the liquid cell extract solution at room temperature. Its protein synthesis capacity will decrease over time until it becomes inactive[4]
[0006] However, there are still no successful cases of freeze-drying other eukaryotic cells, such as yeast, cell-free expression systems
[0007] At present, the freeze-drying process of the cell extract

Method used

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  • Cell-free freeze-drying preparation for in-vitro protein synthesis, and preparation method and application of cell-free freeze-drying preparation
  • Cell-free freeze-drying preparation for in-vitro protein synthesis, and preparation method and application of cell-free freeze-drying preparation
  • Cell-free freeze-drying preparation for in-vitro protein synthesis, and preparation method and application of cell-free freeze-drying preparation

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preparation example Construction

[0210] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0211] (i) providing yeast cells;

[0212] (ii) washing the yeast cells to obtain washed yeast cells;

[0213] (iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0214] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0215] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0216] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0217] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000g, preferably 8000-30000g.

[0218] In the present invention, the centrifugation time is not particu...

Embodiment 1

[0244] Example 1 Preparation of Yeast Cell Extract by Liquid Nitrogen Fragmentation Method

[0245] 1.1 Primary seed culture: inoculate shake flask culture medium with strains frozen at -80°C, culture at 30°C, 200rpm until logarithmic growth phase.

[0246] 1.2 Secondary seed culture: Take an appropriate amount of primary seed bacterial liquid to inoculate secondary seeds, and cultivate at 30°C and 200rpm until logarithmic growth phase.

[0247] 1.3 Batch culture stage: Inoculate the secondary seed bacterial liquid into the fermenter, culture at 30°C for 10-12 hours, enter the fed-batch culture stage, and collect the cell culture when the OD600 value is 50-55.

[0248] 1.4 Pre-cool the cultured cell culture in the ice-water mixture for 10-30 minutes.

[0249] 1.5 Centrifuge the pre-cooled cell culture in 1.4 in a cryogenic centrifuge under the centrifugation conditions: 3,000g, 10min, 4°C to obtain yeast cells.

[0250] 1.6 Use the pre-cooled Washing buffer to resuspend the ...

Embodiment 2

[0258] Example 2 In vitro protein synthesis system

[0259] 2.1 In vitro protein synthesis system: final concentration of 22mM 4-hydroxyethylpiperazineethanesulfonic acid with a pH of 7-8, 30-150mM potassium acetate, 1.0-5.0mM magnesium acetate, 1.5-4mM nucleoside triphosphate mixture (adenoside Purine nucleoside triphosphate, guanosine triphosphate, cytidine nucleoside triphosphate and uridine nucleoside triphosphate), 0.08-0.24mM 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), 1.7mM dithiothreitol, 10-40mM glucose, 200-400mM starch, 0.002-0.02mg / mL amylase, 20-25mM tripotassium phosphate, 0.027 -0.054 mg / mL T7 RNA polymerase, 1%-4% polyethylene glycol, and finally 50% volume of yeast cell extract solution.

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Abstract

The invention provides a cell-free freeze-drying preparation for in-vitro protein synthesis, and a preparation method and application of the cell-free freeze-drying preparation. Specifically, throughthe cell-free freeze-drying preparation provided by the invention, the synthetic activity of exogenous protein can be significantly improved under the situation that a protective agent does not need to be added.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cell-free freeze-dried preparation for protein synthesis in vitro, its preparation method and application. Background technique [0002] Vacuum freeze-drying technology is to freeze the wet material or solution into a solid state at a lower temperature (-10 ° C ~ -50 ° C), and then sublimate the water in it directly into a gaseous state without going through a liquid state under vacuum (1.3 ~ 13 Pa). , the drying technology that finally dehydrates the material. The whole process of vacuum freeze-drying is divided into pre-freezing, sublimation drying in the first stage and re-drying in the second stage. [0003] At present, the freeze-drying method is an effective and commonly used method to maintain protein activity, which is beneficial to storage and transportation, but there are some potential damages in the freeze-drying process[1]. During the process of pre-cooling, primary ...

Claims

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Application Information

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IPC IPC(8): C12P21/02
CPCC12P21/02
Inventor 郭敏周子鉴于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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