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[Expression of PPDK from Microbispora rosea subsp. aerata in Escherichia coli and its application in pyrosequencing]
Bingjie Zou1, Zhiyao Chen, Guohua Zhou
1School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
Abstract:
Pyruvate phosphate dikinase (PPDK; EC 2.7.9.1) is found in certain microorganisms and plants, and catalyzes the conversion of AMP, PPi and phosphoenolpyruvate (PEP) to ATP, Pi and pyruvate. Using the genomic DNA of Microbispora rosea subsp. aerata as the template, a DNA fragment encoding the gene PPDK was amplified by PCR and inserted into the expression vector pET28a(+), yielding pET28a (+)-PPDK. The E. coli BL21 (DE3) was transformed with the pET28a (+)-PPDK. After inducing with IPTG, the E. coli BL21 (DE3) [pET28a (+)-PPDK] expressed recombinant PPDK fused to an N-terminal sequence of 6-His Tag. The molecular weight of PPDK was estimated to be 101 kD by SDS-PAGE. The PPDK was purified by His * Bind Resin affinity chromatography and ultrafiltration using 10 kD cut-off membrane. The successful application of PPDK in pyrosequencing was also demonstrated.
Insights
Researchers successfully expressed and purified Pyruvate phosphate dikinase (PPDK) from Microbispora rosea. This enzyme, crucial for energy metabolism, was shown to be effective in pyrosequencing applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Pyruvate phosphate dikinase (PPDK) catalyzes a key reaction in energy metabolism, converting AMP, PPi, and PEP to ATP, Pi, and pyruvate.
- PPDK is found in various microorganisms and plants, playing a vital role in their metabolic pathways.
Purpose of the Study:
- To clone, express, and purify Pyruvate phosphate dikinase (PPDK) from Microbispora rosea subsp. aerata.
- To demonstrate the functional application of the purified recombinant PPDK in pyrosequencing.
Main Methods:
- Gene amplification of PPDK using PCR from Microbispora rosea genomic DNA.
- Cloning the PPDK gene into the pET28a(+) expression vector for E. coli transformation.
- Purification of the recombinant PPDK using His-tag affinity chromatography and ultrafiltration.
Main Results:
- Recombinant PPDK, fused with an N-terminal 6-His Tag, was successfully expressed in E. coli BL21 (DE3).
- The molecular weight of the purified PPDK was confirmed to be approximately 101 kD via SDS-PAGE.
- The purified enzyme demonstrated successful application in pyrosequencing.
Conclusions:
- The study successfully established a method for producing functional recombinant PPDK from Microbispora rosea.
- The demonstrated utility of PPDK in pyrosequencing opens potential avenues for its application in molecular biology techniques.
