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Evaluation of Maltose Binding Protein-Tagged hATR Kinase Domain Catalytic Activity with p53 Ser-15 Phosphorylation

Rashmi Bhakuni1, Althaf Shaik2, Sivapriya Kirubakaran1,2

  • 1Discipline of Biological Engineering , Indian Institute of Technology Gandhinagar , Gandhinagar - 382355 , Gujarat , India.

Biochemistry
|November 1, 2018
PubMed

Insights

Researchers successfully expressed and purified a soluble, active kinase domain of ATR (ataxia-telangiectasia and Rad3-related kinase). This breakthrough facilitates the development of targeted ATR inhibitors for cancer therapy.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cancer Research

Background:

  • The DNA damage response (DDR) pathway is crucial for maintaining genomic stability.
  • ATR (ataxia-telangiectasia and Rad3-related kinase) is a key mediator in the DDR pathway.
  • ATR inhibition shows promise in sensitizing tumor cells to combination therapies, but specific inhibitors are scarce.

Purpose of the Study:

  • To express, purify, and biologically characterize the kinase domain of ATR.
  • To facilitate the development of specific ATR kinase inhibitors.
  • To enable structure elucidation studies of the ATR kinase domain.

Main Methods:

  • Bacterial expression of the ATR kinase domain.
  • N-terminal fusion with maltose binding protein (MBP) to enhance solubility.
  • Purification of the MBP-tagged hATR kinase domain (MBP-ATR-6X His).
  • Assessment of catalytic activity via substrate phosphorylation (p53 Ser-15).

Main Results:

  • A soluble and catalytically active MBP-tagged hATR kinase domain was successfully produced.
  • The recombinant protein demonstrated phosphorylation activity on p53 Ser-15.
  • This recombinant protein is suitable for further characterization and structure elucidation.

Conclusions:

  • The bacterial expression and purification of a soluble, active ATR kinase domain is feasible.
  • This recombinant protein serves as a valuable tool for identifying and characterizing ATR kinase inhibitors.
  • The findings pave the way for developing novel targeted cancer therapies.

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