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In-vitro protein synthesis system, method of enhancing protein synthesis efficiency by using system and kit including system

A protein synthesis and synthesis system technology, applied in the biological field, can solve the problems of easy blockage of semi-permeable membrane pores, inability to expand the reaction system, and high cost of semi-permeable membrane dialysis devices

Pending Publication Date: 2020-04-07
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are defects that the semipermeable membrane dialysis device is expensive, the reaction system cannot be expanded, and the reaction product is easy to block the pores of the semipermeable membrane.

Method used

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  • In-vitro protein synthesis system, method of enhancing protein synthesis efficiency by using system and kit including system
  • In-vitro protein synthesis system, method of enhancing protein synthesis efficiency by using system and kit including system
  • In-vitro protein synthesis system, method of enhancing protein synthesis efficiency by using system and kit including system

Examples

Experimental program
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preparation example Construction

[0138] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0139] (i) providing yeast cells;

[0140] (ii) washing the yeast cells to obtain washed yeast cells;

[0141] (iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0142] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0143] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0144] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0145] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000 g, preferably 8000-30000 g.

[0146] In the present invention, the centrifugation time is not parti...

Embodiment 1

[0177] The preparation of embodiment 1 cell extract

[0178] In this example, liquid nitrogen freezing and crushing method was used for the treatment of yeast cells.

[0179] 1.1. Primary seed culture: inoculate the shake flask culture medium with -80 ℃ frozen strains, and cultivate at 30 ℃, 200 rpm until the logarithmic growth phase.

[0180] 1.2. Secondary seed culture: Inoculate an appropriate amount of primary seed bacterial liquid to secondary seeds, and cultivate at 30°C and 200 rpm until the logarithmic growth phase.

[0181] 1.3. Batch culture stage: inoculate the secondary seed bacteria liquid into the fermenter, control the temperature at 30 ºC for 10-12 hours, enter the fed culture stage, and collect the cell culture when the OD600 value is 50-55.

[0182] 1.4. Pre-cool the cultivated cell culture in ice-water mixture for 10-30 min.

[0183] 1.5. Centrifuge the pre-cooled cell culture in 1.4 in a cryogenic centrifuge under the centrifugation conditions: 3,000...

Embodiment 2

[0192] Example 2 Freeze-drying of cell extracts

[0193] 2.1 Place the yeast extract stored at -80 ℃ at room temperature and thaw it;

[0194] 2.2 Weigh each glass bottle or glass plate and make a record;

[0195] 2.3 Divide the thawed yeast extract into glass bottles or glass plates, about 500 microliters of cell extract per glass bottle, and about 5 ml of cell extract per glass plate.

[0196] 2.4 Weigh the glass bottle and glass plate containing the cell extract in 2.3 again, and make a record;

[0197] 2.5 Pre-freeze the glass bottle and glass plate containing the cell extract in 2.4 at -80 ℃ for 2-4 hours;

[0198] 2.6 Set the freeze-drying program: pre-freezing stage: -55 °C, 4 h; first freeze-drying stage: -30 °C, 4-10 h; second freeze-drying stage: -30 °C, 10 h; final freeze-drying stage: -20°C, 10-20 h.

[0199] 2.7 Place the pre-frozen cell extract glass bottle and glass plate in 2.5 on the plate of the lyophilizer, and carry out the lyophilization procedure with...

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Abstract

The invention provides an in vitro protein synthesis system, a kit and a preparation method. The in vitro protein synthesis system includes: (a) a cell extract; (b) an adsorbent used for enhancing protein synthesis efficiency; (c) amino acids; (d) nothing or NTPs; and (e) exogenous DNA molecules. A method for enhancing in vitro protein synthesis efficiency is provided. The method adopts the synthesis system and includes the following steps: (1) performing mixing; (2) adding the exogenous DNA molecules; (3) performing incubation synthesis; and (4) performing post-processing; and the method alsoincludes a step of adding the adsorbent after the step (1) or (2). In addition, the kit of the in vitro protein synthesis system is also provided. Therefore, the synthesis efficiency of foreign protein can be significantly enhanced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to an in vitro protein synthesis system and its method and kit for improving protein synthesis efficiency. Background technique [0002] Biological reactions, namely biochemical reactions, refer to chemical reactions carried out in living organisms. These reactions are catalyzed by enzymes. Enzymes and reactants are dissolved in water in the internal environment to react. Water provides carriers and media for substances in the body[1] . In the last few decades of the 20th century, biochemistry has made great achievements in explaining life processes, and now almost botany, medicine, genetics and other life science-related fields are engaged in biochemical research [2]. [0003] The traditional protein expression system refers to a multi-step cascade biochemical reaction technique for expressing foreign genes through model organisms, including bacteria, fungi, pla...

Claims

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Application Information

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IPC IPC(8): C12N15/63
CPCC12N15/63
Inventor 郭敏徐开宋亚玲李海洋于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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