Identification method of intracellular and extracellular proteomes of penicillium during fermentation

A technology of proteome and identification method, applied in the field of protein identification to achieve the effect of improving yield

Inactive Publication Date: 2013-09-18
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, except for penicillin synthesis genes, metabolic engineering, and microproteomics analysis, few studies have been conducted on the intracytoplasmic and extracellular proteome of this fungus, especially under industrial fermentation conditions that may be directly or indirectly involved in penicillin biosynthesis. Happening

Method used

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  • Identification method of intracellular and extracellular proteomes of penicillium during fermentation
  • Identification method of intracellular and extracellular proteomes of penicillium during fermentation
  • Identification method of intracellular and extracellular proteomes of penicillium during fermentation

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Extraction of extracellular and extracellular proteome of Penicillium during fermentation

[0036] (1) Extraction of intracellular protein:

[0037] Penicillium chrysogenum (Penicillium chrysogenumCGMCC NO.3.4023) was inoculated in the LB medium (this embodiment adopts Penicillium chrysogenum CGMCC NO.3.4023)), cultivated, from 2h to 170h after inoculation, 10 parts of cells were collected at 10 time points (respectively at 2h , 8h, 20h, 32h, 50h, 80h, 98h, 128h, 140h and 170h), each cell was washed three times with 2mM phenylmethylsulfonyl fluoride-ultrapure aqueous solution in ice bath; Grind the cells into a cell powder in liquid nitrogen; take 0.2 g of the cell powder and suspend it in the lysis buffer, then add 12 μl of aqueous solution of DNase I and RNaseA, place it in an ice bath for 20 min, add 12 μl of 1 mM phenylmethylsulfonate Acylated fluorine-isopropanol solution, ultrasonic 15 seconds / time, 3 times in total, centrifuged at 20,000xg for 40 minutes, collec...

Embodiment 2

[0044] A method for identifying the intracellular and extracellular proteomes of Penicillium during fermentation, comprising the steps of:

[0045] (1) Two-dimensional gel electrophoresis

[0046] ①Isoelectric focusing electrophoresis

[0047] Dissolve 800 μg of dried protein samples (extracted in Example 1) from the inside and outside of Penicillium cells with 350 μL of swelling solution (rehydration buffer), focus the protein with an IPGphor instrument, and the conditions of isoelectric focusing electrophoresis: 30V electrophoresis for 13 hours, 500V Electrophoresis 1h, 1000V electrophoresis 1h, 8000V electrophoresis 10h;

[0048] ②Balance of IPG strips:

[0049] After isoelectric focusing electrophoresis, put the IPG gel strip into a glass tube containing 10ml SDS balance solution I and shake it on the shaker for 15min, then transfer the strip to a glass tube containing 10ml SDS balance solution II and shake it. Shake and balance on the bed for 15 minutes;

[0050] ③SDS-P...

Embodiment 3

[0059] A method for identifying the intracellular and extracellular proteomes of Penicillium during fermentation, comprising the steps of:

[0060] (1) Two-dimensional gel electrophoresis

[0061] ①Isoelectric focusing electrophoresis

[0062] Dissolve 800 μg of dried protein samples (example 1) extracted from the inside and outside of Penicillium cells with 300 μL of swelling solution (rehydration buffer), focus the protein with an IPGphor instrument, and perform isoelectric focusing electrophoresis conditions: 30V electrophoresis for 15 hours, 500V Electrophoresis 3h, 1000V electrophoresis 3h, 8000V electrophoresis 9h;

[0063] ②Balance of IPG strips:

[0064] After the isoelectric focusing is over, put the IPG strip into a glass tube containing 5ml SDS balance solution I and shake it on the shaker for 10min, then transfer the strip to a glass tube containing 5ml SDS balance solution II and place it on the shaker Shake and balance for 10 minutes;

[0065] ③SDS-PAGE elect...

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Abstract

The invention discloses an identification method of intracellular and extracellular proteomes of penicillium during fermentation. The identification method comprises the following steps of: (1) extracting intracytoplasmic proteins; (2) extracting extracytoplasmic proteins; (3) carrying out two-dimensional gel electrophoresis; (4) analyzing a two-dimensional gel image; and (5) identifying the proteins by using MALDI (Matrix-Assisted Laser Desorption Ionization)-TOF (Time-Of-Flight)-MS (Mass Spectrometry). The protein level closely related to penicillin synthesis in a penicillin fermentation process, and a relation between the protein level and the penicillin production level can be found quickly and accurately in high flux by using the identification method, differentially expressed proteins are found out and provide a target spot for further modifying a penicillium metabolic path, thus a certain theory is provided for obtaining high production strains and improving the yield of penicillin, and a new method and a concept are provided for modifying other industrial antibiotic strains.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and relates to a novel method for identifying proteins in cells and in fermentation medium during fermentation. Background technique [0002] As one of the largest global sales of antibiotic drugs, Penicillium chrysogenum for industrial fermentation production has undergone several strain improvements, and the biosynthetic pathway of penicillin has been clearly described. The availability of the gene sequence of Penicillium chrysogenum now makes it possible to study penicillin G production at the proteomic level. However, except for penicillin synthesis genes, metabolic engineering, and microproteomics analysis, few studies have been conducted on the intracytoplasmic and extracellular proteome of this fungus, especially under industrial fermentation conditions that may be directly or indirectly involved in penicillin biosynthesis. Condition. It is reported that autolysis leads to the de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447G01N27/62
Inventor 元英进程景胜乔斌吕晓敏卢华陈尧
Owner TIANJIN UNIV
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