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Method for preparing cephalosporium acremonium proteome

A technology of Cephalosporium acremonium and proteome, applied in the biological field, can solve the problems of no Cephalosporium acremonium, no unified protein extraction standard, etc., and achieve the effect of optimizing metabolic engineering

Inactive Publication Date: 2012-05-09
SHANGHAI INST OF PHARMA IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no proteomic studies on Cephalosporium acremonium
[0003] Proteome preparation is the key to comparative proteomics technology, and there is no uniform standard for protein extraction

Method used

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  • Method for preparing cephalosporium acremonium proteome
  • Method for preparing cephalosporium acremonium proteome
  • Method for preparing cephalosporium acremonium proteome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Extraction of Cephalosporium acremonium proteome sample

[0052] 1. Incline culture and strain preservation of Acremonium chrysogenum 46117

[0053] Inoculate Acremonium chrysogenum ATCC46117, the CPC-producing bacterium of Cephalosporium acremonium, on the slant surface of the moist basal medium (wort juice 20%, maltose 4.0, polyptone 1%, pH 7.0, plus purified agar powder 2%), and cultured at 28°C for 10 ~14d (days), until full spores grow. The grown slant can be stored in a refrigerator at 4°C for about 1 month. For long-term preservation, add an equal volume of 40% sterile glycerin to the arthrospore suspension, mix well, freeze with liquid nitrogen and quickly store at -70°C. For long-term storage, mix arthrospores with skimmed milk, pack them into frozen glass tubes, freeze-dry them with a freeze dryer, and finally vacuum seal them and store them at 4°C.

[0054] 2. Fermentation of Acremonium chrysogenum ATCC 46117

[0055] Scrape an appropriate amoun...

Embodiment 2

[0066] Example 2 Separation of proteome samples by two-dimensional electrophoresis

[0067] Take a total of 1mg of protein samples and mix them with urea hydration solution (8M urea, 2% CHAPS, 0.5% IPG buffer, 18mM DTT, 0.002% bromophenol blue) to form a total of 450μl of sample solution, and place it at room temperature for 1h (25°C) with gentle shaking. Put the hydrated protein solution into the DryStrip foaming tray, drag the 24cm Immobiline DryStrip dry strip to soak the strip evenly in the protein solution, and soak for 12-16 hours (25°C). Transfer the foamed strip to the Manifold strip slot, and use the Ettan IPGphor III system for isoelectric focusing. The focusing program is (24cm strip) 50V 4h, 100V 3h, 1000V 2h, 3000V 2h, 5000V 2h, 10000V 10h , the upper limit current is 50μA / strip, and the focusing temperature is 20°C. After focusing, prepare SDS balance solution (6M urea, 75mMTris-Cl pH 8.8, 29.3% glycerol, 2% SDS, 0.002% bromophenol blue), add 10ml / strip balance...

Embodiment 3

[0069] Example 3 Detection of proteome under different conditions of two-dimensional electrophoresis

[0070] The first direction of two-dimensional electrophoresis is isoelectric focusing. According to the different isoelectric points of various proteins and the characteristic of converging toward their isoelectric points, proteins can be separated along the gradient to their respective isoelectric points. The second dimension is SDS-polyacrylamide gel electrophoresis, which separates proteins and peptides according to their molecular weight.

[0071] The gel strips commonly used to separate proteins are divided into linear and nonlinear, and their pH ranges are different. The difference between linear and nonlinear strips is that the isoelectric points of linear strips are equidistantly distributed, while the distance between 4-7 is extended for non-linear strips. Broad-range strips can be used to get a rough overview of protein distribution, while narrow-range strips can b...

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Abstract

The invention discloses a method for preparing cephalosporium acremonium proteome, which comprises the following steps that: cephalosporium acremonium thalli are ground into powder with liquid nitrogen, and then, total protein samples are extracted by a trichloroacetic acid (TCA)-acetone precipitation method; the obtained total protein samples are mixed with hydrated solution, hydration sampling is carried out, immobilized pH gradient (IPG) solid phase rubber strips are adopted, next, isoelectric focusing is carried out, the rubber strips are balanced after the isoelectric focusing completion and are then transferred onto sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, electrophoresis is carried out, the electrophoresis is stopped when bromophenol blue indicators reach the bottom edge, and the gel is obtained; and the transmission scanning is carried out with a gel scanning instrument after the obtained gel is dyed, and the obtained images are analyzed by using software. The cephalosporin C has the important place in the cephalosporin antibiotics production, so the study on the cephalosporium acremonium proteome can be carried out, the foundation is laid for optimizing the molecular breeding and the metabolic engineering of cephalosporin producing strains, and the important significance is realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a preparation method of Cephalosporium acremonium proteome. Background technique [0002] Cephalosporin acremonium is the main industrial strain used to produce cephalosporin C by fermentation, and cephalosporin C is the precursor of 7-aminoalkanoic acid, a key intermediate of cephalosporin antibiotics. Comparative proteomics is an important field of proteomics research, which is used to analyze the changes and differences of proteomes under different conditions, and to discover and identify differential proteins in different physiological states. In view of the important position of cephalosporin C in the production of cephalosporin antibiotics, comparative proteomics research on Cephalosporium acremonium was carried out to find the difference in the protein level between high-yielding bacteria and low-yielding bacteria, so as to optimize cephalosporins through genetic engineering. ...

Claims

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Application Information

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IPC IPC(8): C07K1/36C07K1/30C07K1/28C07K1/26
Inventor 胡又佳刘艳朱春宝朱宝泉刘红龚桂花
Owner SHANGHAI INST OF PHARMA IND
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