Heterologous Expression, Purification and Enzymatic Activity of Cyclic Nucleotide Phosphodiesterase 1 from Saccharomyces cerevisiae

doi:10.7506/spkx1002-6630-20190711-161

FOOD SCIENCE ›› 2020, Vol. 41 ›› Issue (18): 71-76.doi: 10.7506/spkx1002-6630-20190711-161

• Bioengineering • Previous Articles Next Articles

Heterologous Expression, Purification and Enzymatic Activity of Cyclic Nucleotide Phosphodiesterase 1 from Saccharomyces cerevisiae

CHEN Ying, LI Chixia, CHEN Yujuan, TIAN Yuanyuan, ZHANG Meng, HAN Yingyi, WANG Yousheng

(Beijing Advanced Innovation Center for Food Nutrition and Human Health, School of Light Industry, Beijing Technology & Business University (BTBU), Beijing 100048, China)

Online:2020-09-25 Published:2020-09-18

Abstract

Abstract: In order to prepare high-purity cyclic nucleotide phosphodiesterase 1 (PDE1) from Saccharomyces cerevisiae, the pGM-T-S. cerevisiae was used as a template to amplify the target gene by PCR, and an expression plasmid carrying this gene was constructed, and then transformed into Escherichia coli BL21 cells for expression. High-pressure broken bacteria extract was purified consecutively by nickel affinity column chromatography, Q-Sepharose ion exchange column chromatography and Sephacryl S200 column chromatography. The purified enzyme activity remained 92% of the original activity. This study may provide a basis for future crystallographic analysis of the protein and its metabolic regulation in S. cerevisiae.

Key words: Saccharomyces cerevisiae; cyclic nucleotide phosphodiesterase; expression and purification; enzyme activity determination

CLC Number:

TS201.2

CHEN Ying, LI Chixia, CHEN Yujuan, TIAN Yuanyuan, ZHANG Meng, HAN Yingyi, WANG Yousheng. Heterologous Expression, Purification and Enzymatic Activity of Cyclic Nucleotide Phosphodiesterase 1 from Saccharomyces cerevisiae[J]. FOOD SCIENCE, 2020, 41(18): 71-76.

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Heterologous Expression, Purification and Enzymatic Activity of Cyclic Nucleotide Phosphodiesterase 1 from Saccharomyces cerevisiae

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