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Method for extracting fungus total protein

An extraction method and technology of total protein, which are applied in the fields of peptide preparation, chemical instruments and methods, organic chemistry, etc., can solve the problems of lack of general methods, etc., and achieve the effect of great practical application value.

Inactive Publication Date: 2013-09-25
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, people's research on fungi is mostly concentrated on some fungi that have application value or have pathogenic effects on humans. Most of the methods are also aimed at a certain strain or a certain type of strain, and there is a lack of a general method.

Method used

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  • Method for extracting fungus total protein
  • Method for extracting fungus total protein
  • Method for extracting fungus total protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Five kinds of methods and the method of the present invention extract the one-way comparative experiment of Mortierella flavinum total protein effect

[0028] After searching for information, five methods were selected to extract the total protein of Mortierella chrysogenum, and SDS-PAGE was used to compare the pros and cons of the extraction methods. The specific methods include the following steps:

[0029] 1. Bacteria culture

[0030] Pick the spores of Mortierella chrysogenum on the slant in PDA liquid medium, and cultivate them at 28°C and 150rmp for 72h;

[0031] 2. Five methods are used to extract the total protein of Mortierella flavinum. The five methods are as follows:

[0032] Method 1: Collect the cultured bacteria by suction filtration, wash with ultrapure water for 3 times, and then collect by filtration. A part of the collected bacteria is fully ground in liquid nitrogen, and 100 mg of ground mycelium powder is taken Put 1ml of lysate (20 ...

Embodiment 2

[0053] Embodiment 2: Under different extraction conditions, adopt the method of the present invention to carry out the experiment of total protein extraction to Mortierella flavinum

[0054] (1) The extraction method is the same as in Example 1, except that the extraction solution I is 7.5M urea, 2.5M thiourea, 1.5mM EDTA, 10mM Tris-HCL (pH7.4), 10ul protease inhibitor mixture, and the incubation time is 50min , mix once every 6 minutes; extract II is 2% CHAPS, 0.5% SDS, 2% DTT, incubate for 25 minutes, mix once every 4 minutes during the incubation;

[0055] (2) The extraction method is the same as in Example 1, except that the extraction solution I is 7M urea, 2M thiourea, 1mM EDTA (ethylenediaminetetraacetic acid), 10mM Tris-HCL (trishydroxymethylaminomethane hydrochloride, pH7 .4), 10ul protease inhibitor mixture, the incubation time is 40min, mixed every 7min; the extract II is 2% CHAPS (3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid Salt), 0.5% SDS (sodium dod...

Embodiment 3

[0062] Embodiment 3: the comparison of different bacterium powder addition amounts to extraction effect

[0063] Add 100 mg, 110 mg, 120 mg, 130 mg, 130 mg, and 150 mg of bacterial cell powder respectively according to the method of the present invention in Example 1 to compare the total protein concentration, and the results are shown in Table 4.

[0064] Table 4: Comparison of the extraction effect of different bacterial powder additions

[0065]

[0066] It can be seen from the table that the addition of bacterial cell powder cannot significantly increase the total protein concentration.

[0067]

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Abstract

The invention provides a method for extracting fungus total protein. A simple and feasible method is adopted, the characteristics that the fungal cell wall is thick and tough and the intracellular protein is difficult to release are overcome, and high-quality fungus proteome is successfully extracted. The components of an extracting solution are simple; the extracting solution I comprises 7-8M urea, 2-2.5M thiourea, 1-2m MEDTA (ethylenediamine tetraacetic acid), 10mMTris-HCL (trihydroxy methyl-aminomethane hydrochloride with the pH of 7.4) and 10ul protease inhibitor mixture; the extracting solution II comprises 2-4 percent of CHAPS (3-[3-(cholylaminopropyl) dimethylamino]CHAPS, 0.5 percent of SDS (sodium dodecyl sulfate) and 2-4 percent of DTT (dithiothreitol). The method is simple, convenient and rapid and is low in equipment requirement; and moreover, the extraction process of total protein can be finished within three hours, lots of total protein can be obtained and is used for proteome analysis, and the method has high actual application value.

Description

technical field [0001] The invention relates to a method for extracting total protein in the field of proteomics research, in particular to an efficient and rapid method for extracting total protein of fungi. Background technique [0002] Fungi (Fungus) belong to the fungal superclass in algae plants in biological classification, and are microorganisms with eukaryotic cell types. With the deepening of human research on fungi, fungi have been closely related to human life. Fungi are widely distributed in nature, and most of them are beneficial to people, such as wine making, sauce making, fermented feed, farmland fertilization, antibiotic production, mushroom production, food processing and providing Chinese herbal medicine sources (such as Ganoderma lucidum, Poria cocos, Cordyceps, etc., are all products of fungi either themselves or prepared by the action of fungi). However, there are also fungi that are pathogenic to humans, which are divided into superficial fungi and d...

Claims

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Application Information

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IPC IPC(8): C07K1/14
Inventor 张琦胡彬彬何仕武魏云林林连兵季秀玲
Owner KUNMING UNIV OF SCI & TECH
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