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Updated: Aug 30, 2026
Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
Published on: August 29, 2025
Purification and characterization of human nucleolar phosphoprotein 140 expressed in Escherichia coli
Yun-Kyeong Kim1, Youngnam Jin, Krishna M Vukoti
1Division of Life Sciences, Korea Institute of Science and Technology, 39-1, Hwawolkok-dong, Songbuk-ku, 136-791 Seoul, South Korea.
Abstract:
Human nucleolar phosphoprotein 140, hNopp140, is one of the most highly phosphorylated mammalian proteins, which is involved in the biogenesis of nucleolus. It regulates the transcription of rDNA and has a tendency to bind to doxorubicin, which is widely used as an anti-cancer drug. The biochemical and biophysical property of hNopp140 has not been reported due to the fact that it is rather difficult to obtain protein in large enough quantity. In this paper, we report the cloning and overexpression of the soluble form of hNopp140 in Escherichia coli. The protein was purified to more than 90% homogeneity using hydroxyapatite and ion exchange chromatography. The purified protein can be extensively phosphorylated by casein kinase II and oligomerized into an insoluble aggregate in the presence of magnesium, carbonate, and fluoride ions.
Insights
Researchers cloned and overexpressed human nucleolar phosphoprotein 140 (hNopp140), a key protein in nucleolus biogenesis. The purified protein can be phosphorylated and forms aggregates under specific conditions.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Human nucleolar phosphoprotein 140 (hNopp140) is a highly phosphorylated protein crucial for nucleolus biogenesis.
- hNopp140 regulates rDNA transcription and interacts with the anti-cancer drug doxorubicin.
- Previous studies were limited by difficulties in obtaining sufficient quantities of hNopp140 for biochemical and biophysical analysis.
Purpose of the Study:
- To clone and overexpress a soluble form of hNopp140 in Escherichia coli.
- To purify the recombinant hNopp140 protein.
- To investigate the biochemical properties of purified hNopp140, including phosphorylation and aggregation.
Main Methods:
- Cloning and overexpression of soluble hNopp140 in E. coli.
- Purification using hydroxyapatite and ion exchange chromatography.
- Phosphorylation assays using casein kinase II and aggregation studies with magnesium, carbonate, and fluoride ions.
Main Results:
- Successfully cloned and overexpressed soluble hNopp140 in E. coli.
- Purified hNopp140 to over 90% homogeneity.
- Demonstrated that purified hNopp140 can be phosphorylated by casein kinase II and oligomerizes in the presence of specific ions.
Conclusions:
- The study provides a method for obtaining large quantities of purified hNopp140.
- The findings offer insights into the biochemical properties of hNopp140, including its phosphorylation and aggregation behavior.
- This work facilitates further investigation into hNopp140's role in nucleolar function and its potential interactions with therapeutic agents.
