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A protein synthesis system, kit and preparation method thereof for in vitro protein synthesis

A protein synthesis and protein technology, used in biological testing, microbial dissolution, instruments, etc., can solve the problems of high cost, high cost, and no eukaryotic in vitro protein expression system, and achieve the effect of low cost and high yield

Active Publication Date: 2019-04-19
KANGMA SHANGHAI BIOTECH LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the prokaryotic system, the cultivation of eukaryotic cells is difficult and expensive, and the preparation process of the cell extract is cumbersome, so their translation system is relatively expensive and only suitable for special laboratory use
Therefore, eukaryotic in vitro protein expression systems suitable for industrial large-scale (ton-level) preparation and production do not currently exist

Method used

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  • A protein synthesis system, kit and preparation method thereof for in vitro protein synthesis
  • A protein synthesis system, kit and preparation method thereof for in vitro protein synthesis
  • A protein synthesis system, kit and preparation method thereof for in vitro protein synthesis

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preparation example Construction

[0132] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0133] (i) providing yeast cells;

[0134] (ii) washing the yeast cells to obtain washed yeast cells;

[0135] (iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0136] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0137] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0138] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0139] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000×g, preferably 8000-30000×g.

[0140] In the present invention, the centrifugation time is not parti...

Embodiment 1

[0171] Embodiment 1: high pressure crushing prepares yeast cell extract

[0172] 1.1 Preparation of yeast seed liquid: Pick a single colony of Kluyveromyces lactis from the plate and inoculate it in 50 mL of YPD medium (the composition of YPD medium is: 1% yeast extract, 2% peptone, 2% glucose, pH 5.5) in a 250mL Erlenmeyer flask (the liquid content is 20%, the same below), and the inoculated Erlenmeyer flask was placed in a shaker for cultivation. What is obtained after h is the seed liquid;

[0173] 1.2 Yeast cell culture: Inoculate the seed liquid prepared in 1.1 into a 2 L Erlenmeyer flask containing 400 mL of YPD medium according to the inoculation amount of 0.1-1 %, and place it in a shaker for culture. The culture condition: the temperature is 30 °C, the rotation speed is 200rpm. In the middle and late stages of the logarithmic phase of yeast growth (OD600=3.0-6.9), the culture is terminated to obtain cell culture fluid;

[0174] 1.3 Pre-cool the cell culture culture...

Embodiment 2

[0184] Embodiment 2: liquid nitrogen crushing method prepares yeast cell extract

[0185] 2.1 Preparation of yeast seed liquid: Pick a single colony of Kluyveromyces lactis from the plate and inoculate it in 50 mL of YPD medium (the composition of YPD medium is: 1% yeast extract, 2% peptone, 2% glucose, pH 5.5) in a 250mL Erlenmeyer flask (filling volume is 20%, the same below), and the inoculated Erlenmeyer flask was placed in a shaker for culture, culture conditions: temperature 30 ℃, rotation speed 200 rpm. After culturing for 24 h, the seed solution was obtained;

[0186] 2.2 Yeast cell culture: Inoculate the seed solution prepared in 2.1 into a 2 L Erlenmeyer flask containing 400 mL of YPD medium according to the inoculum amount of 0.1-1 %, and place it in a shaker for culture. The culture condition: the temperature is 30 °C, the rotation speed is 200rpm. In the middle and late stages of the logarithmic phase of yeast growth (OD600=3.0-6.9), the culture is terminated to...

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Abstract

The present invention provides a protein synthesis system, kit and preparation method for in vitro protein synthesis. Specifically, the in vitro cell-free expression system provided by the present invention can not only synthesize proteins extremely efficiently, but also synthesize complex proteins, and Using the in vitro cell-free expression system of the present invention, the relative light unit value of the luciferase activity synthesized is at least one order of magnitude (≥10 times or higher) higher than that of the current commercial system (such as the rabbit reticulocyte in vitro expression system).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein synthesis system, kit and preparation method for in vitro protein synthesis. Background technique [0002] The traditional protein expression 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, a cell-free expression system, also known as an in vitro protein synthesis system, emerges as the times require, which uses an exogenous target mRNA or DNA as a template for protein synthesis, and artificially controls the addition of substrates and transcription required for protein synthesis. Substances such as translation-related protein factors can realize the synthesis of the target protein. Expressing proteins in an in vitro translation system does not require the steps of plasmid construction, transformation, cell culture, cell ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/6812C12N1/06C12P21/00G01N33/68
Inventor 郭敏柴智刘帅龙符雷王海鹏于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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