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Method for screening corynebacterium crenatum endogenous high-expression promoter by using 2-DE (Two-Dimensional Electrophoresis) technique

A Corynebacterium blunt tooth, 2-DE technology, applied in the field of proteomics and genetic engineering, can solve the problems of application limitations, low efficiency of exogenous promoters, etc.

Active Publication Date: 2013-05-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the widely used corynebacterium expression vectors mostly use exogenous promoters such as P-tac and lacZ from Escherichia coli. The efficiency of these exogenous promoters is generally not high, and their application is limited by many factors.

Method used

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  • Method for screening corynebacterium crenatum endogenous high-expression promoter by using 2-DE (Two-Dimensional Electrophoresis) technique
  • Method for screening corynebacterium crenatum endogenous high-expression promoter by using 2-DE (Two-Dimensional Electrophoresis) technique
  • Method for screening corynebacterium crenatum endogenous high-expression promoter by using 2-DE (Two-Dimensional Electrophoresis) technique

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: According to 2-DE screening corynebacterium bacillus endogenous highly active promoter

[0035] After culture and protein extraction, the extracted protein concentration is about 70 μg / μL. After the first dimension isoelectric focusing, equilibration and second dimension SDS-PAGE, after fixation, staining and decolorization, a condensate with clear protein distribution was obtained. Gel; use Lad Scan (GE Health) to scan the gel, and then use ImageMaster 2D Platinum5.0 software to adjust, find, quantify and match the gel image, and cut the selected protein spots for MALDI-TOF-MS And MS / MS analysis, a total of 15 highly expressed protein spots were successfully identified.

Embodiment 2

[0036] Embodiment 2: Construction of recombinant expression vector and bacterial strain

[0037] Based on the 15 highly expressed protein spots obtained in Example 1, combined with information such as their copy numbers in bacterial cells, 5 target protein spots were found, and the gene sequences corresponding to these 5 proteins were found in GENBANK , and separated its promoter sequence according to the gene sequence, and named these five promoters as P-argC, P-argG, P-argF, P-ilvC and P-serA. These five promoters were respectively replaced with the tac promoter on the shuttle vector pDXW-8, and the reporter gene chloramphenicol acetyltransferase gene cat gene was inserted downstream of it, and the recombinant vectors were transformed into Escherichia coli JM109 and Corynebacterium bacilli . After verification, the successful construction of pDXW-P argC -cat, pDXW-P argG -cat, pDXW-P argF -cat, pDXW-P ilvC -cat and pDXW-P serA -cat, and successfully obtained its corres...

Embodiment 3

[0038] Embodiment 3: the enzyme activity assay of recombinant bacterial strain

[0039] The recombinant strains were cultured in LB and LBG medium respectively, centrifuged at 8000rpm for 10min, washed twice with 100mM Tris-HCl buffer solution of pH 7.8, suspended in the buffer solution, and subjected to ultrasonic crushing to prepare crude enzyme solution. The reaction mixture is composed of a certain volume of crude enzyme solution, 100mM Tris-HCL (pH7.8), 0.1mM acetyl-CoA, and 0.4mg / mL DTNB, reacted at 37°C for 2min, added chloramphenicol to terminate the reaction, and detected Changes in absorbance at 412nm. The enzyme activity unit is defined as the amount of enzyme required to acetylate 1 μmol of chloramphenicol per minute.

[0040] The enzyme activity of the recombinant Escherichia coli was measured as image 3 Respectively: E.coli JM109 / pDXW-P argC -cat is 0.36U / mg, E.coli JM109 / pDXW-P argG -cat is 5.58U / mg, E.coli JM109 / pDXW-P argF -cat is 3.81U / mg, E.coli JM109 / ...

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Abstract

The invention discloses a method for screening a corynebacterium crenatum endogenous high-expression promoter by using a 2-DE (Two-Dimensional Electrophoresis) technique, which belongs to the fields of proteomics and gene engineering. The method comprises the following steps of: identifying a high-expression protein point in a corynebacterium crenatum mycetocyte through the 2-DE technique in combination with a mass-spectrometric technique; and cloning encoding gene promoters, naming as P-argC, P-argG, P-argF, P-ilvC and P-serA respectively, replacing a tac promoter on a shuttle vector pDXW-8, inserting a cat gene downstream, transforming colon bacillus JM109 and corynebacterium crenatum SYPA5-5 respectively, and measuring the activity of CAT (Catalase), wherein the activities of the prompters P-argG, P-argF, P-ilvC, P-serA and P-argC in a recombinant strain decrease in sequence. According to the method, a constitutive high-expression promoter from corynebacterium crenatum is obtained for the first time, and an effective tool is provided for a corynebacterium crenatum high-expression exogenous gene.

Description

technical field [0001] The invention relates to a method for screening endogenous high-expression promoters of Corynebacterium blunt-toothed by 2-DE technology, which belongs to the field of proteomics and genetic engineering. technical background [0002] Two-dimensional electrophoresis (2-DE) is the core technology of proteome research. 2-DE technology was independently established by O Farrell and Klose in two laboratories in 1975. They combined high-resolution isoelectric focusing (Iso-electric focusing, IEF) electrophoresis and sodium dodecylsulfonate-polypropylene Amide gel electrophoresis (SDS-PAGE) combined with two-dimensional electrophoresis. The basic principle is: in the first direction, isoelectric focusing isoelectric focusing in the pH gradient gel based on the difference in the isoelectric point of the protein; in the second direction, the separation is carried out according to the difference in molecular weight along the direction perpendicular to the first...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/77C12N1/21C12R1/15
Inventor 饶志明许正宏张博慧徐美娟
Owner JIANGNAN UNIV
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