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Kit for in vitro synthesis of protein and preparation method

A technology of protein synthesis and protein synthesis, which is applied in the field of yeast cell extract kits, can solve the problems of non-existence and achieve the effects of convenient transportation, convenient operation and rapid reproduction

Active Publication Date: 2017-07-25
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, no Kluyveromyces in vitro protein synthesis system exists

Method used

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  • Kit for in vitro synthesis of protein and preparation method
  • Kit for in vitro synthesis of protein and preparation method
  • Kit for in vitro synthesis of protein and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: High-pressure preparation of cell extracts for in vitro protein synthesis

[0043] 1.1 Pick a single colony of Kluyveromyces lactis (strain number ATCC 8585) and inoculate it in 50 mL of YPD medium (filling volume is 20%, the same below). The composition of YPD medium is: 1% yeast extract , 2% peptone, 2% glucose, 30 ℃, 200 rpm, cultivated for 24 h;

[0044] 1.2 Inoculate the above bacterial liquid into 500 mL of YPD medium according to the inoculum amount of 1%, harvest the yeast cells by centrifugation at 30 °C, 200 rpm, when OD600=3.0-6.9, and 4 °C;

[0045] 1.3 The above bacteria were resuspended in pre-cooled Buffer A for 3 times. The composition of Buffer A is: 25 mM potassium 4-hydroxyethylpiperazine ethanesulfonate pH 7.4, 150 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride;

[0046] 1.4 High-pressure crushing, the pressure value is 1400 bar, and the ratio of bacteria to Buffer A is 2 g: 1 mL...

Embodiment 2

[0050] Example 2: Preparation method of liquid nitrogen in agitator for in vitro protein synthesis cell extract

[0051] 2.1 Pick a single colony of Kluyveromyces lactis (strain number ATCC 8585) and inoculate it in 50 mL of YPD medium (filling volume is 20%, the same below). The composition of YPD medium is: 1% yeast extract , 2% peptone, 2% glucose, 30 ℃, 200 rpm, cultivated for 24 h;

[0052] 2.2 Inoculate the above bacterial solution into 500 mL of YPD medium according to the inoculum amount of 1%, at 30 ℃, 200 rpm, when OD600=3.0-6.9, and harvest the yeast cells by centrifugation at 4 ℃;

[0053] 2.3 The above bacteria were resuspended in pre-cooled Buffer A for 3 times. The composition of Buffer A is: 25 mM potassium 4-hydroxyethylpiperazine ethanesulfonate pH 7.4, 150 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride;

[0054] 2.4 Use a blender (electronic extractor, juicer) to crush with liquid nitrogen, add an ...

Embodiment 3

[0058] Embodiment 3: the lyophilization preservation method of in vitro protein synthesis cell extract

[0059] 3.1 The solutions obtained in Example 1.6 and Example 2.6 were divided into 0.5-10 mL and quick-frozen in liquid nitrogen;

[0060] 3.2 Freeze drying using a lyophilizer. After the sample is made into a dry powder (<5% moisture), store in airtight low temperature or room temperature;

[0061] 3.3 For freeze-dried samples, add water to the same volume as before freeze-drying;

[0062] 3.4 After shaking to dissolve, it can be used.

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Abstract

The invention provides a preparation method of a yeast cell extracting solution, the yeast cell extracting solution and a method for synthesizing protein in an in vitro system by virtue of the yeast cell extracting solution, as well as a kit containing the yeast cell extracting solution. The kit, which is more convenient than a conventional method, is applicable to protein synthesis in the in vitro system; transformation, culture and crushing can be prevented; a great amount of using time and cost can be saved; various types of proteins can be expressed, and influence of protein toxicity can be prevented; and a plurality of protein complexes can be expressed, without the use of a high temperature at 37 DEG C. In addition, as a raw material adopted by the invention, yeast cell is simple to cultivate, convenient to operate, rapid to propagate and relatively low in cost; a prepared yeast extract has a capacity of modifying translated protein, the yeast extract is suitable for large-scale preparation and has an advantage of industrial production; and the yeast cell extract, which is prepared into freeze-dried powder by virtue of a vacuum freeze-drying method and is preserved at low temperature or high temperature, is convenient to transport.

Description

technical field [0001] The present invention relates to a method for preparing a yeast cell extract used for in vitro protein synthesis, a yeast cell extract and a method for synthesizing proteins in an in vitro system using the yeast cell extract, and the A kit for yeast cell extract, which can be used for protein synthesis in an in vitro system, belongs to the field of biotechnology. Background technique [0002] Traditional protein expression refers to the biological technology of expressing foreign genes through bacteria, fungi, plant cells or animal cells. With the development of science and technology, protein-related frontier fields are gradually emerging, and in vitro protein synthesis systems have emerged, which use exogenous mRNA or DNA as a template to synthesize corresponding mRNA under the action of components such as RNA polymerase and transcription factors. An in vitro system for protein translation by adding amino acids, ATP and GTP to the system [1]. The c...

Claims

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Application Information

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IPC IPC(8): C12N1/06C12P21/00C12R1/645
CPCC12N1/066C12P21/00
Inventor 郭敏符雷柴智刘帅龙于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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