Purification and Characterization of Prolyl Hydroxylase 3/Pyruvate Kinase Isoform 2 Protein Complex

  • Sunil Kumar
  • Ashok Kumar PatelEmail author
Original Paper


The prolyl hydroxylase 3 (PHD3) protein is less abundant in normal oxygen conditions (normoxia) but increases under deficient oxygen condition (hypoxia). Since cancerous cells often thrive in hypoxic conditions and predominantly express the Pyruvate kinase isoforms 2 (PKM2), the PHD3/PKM2 interaction might be particularly important in cancer development. In the present study, the PHD3/PKM2 complex was co-expressed and purified by size-exclusion chromatography. The interaction of PHD3 with PKM2 was confirmed in Native gel as well as western blot analysis. The PHD3/PKM2 complex formed discreet crystals under suitable conditions, and diffraction data revealed that crystal belonged to the P1 space group with 3.0 Å resolution. This is the first crystal report of PHD3/PKM2 complex as well as this study demonstrates a direct physical binding through protein–protein interaction. The structural analysis of complex will provide the information regarding the amino acid residues critical for the catalytic mechanism. Based on the structural information thus obtained, pharmacological interference with the PHD3/PKM2 interaction could be used as a novel strategy to reduce the cancer progression.


PHD3/PKM2 complex Protein crystal Protein interaction Cancer proteins 



We are thankful to the beamline scientists at the European Synchrotron Radiation Facility, Grenoble, France for assisting us with the use of beamline ID30A-3. The authors acknowledge the infrastructural support from Indian Institute of Technology Delhi. SK acknowledges the research grant from SERB, Department of Science & Technology, and Govt. of India. Authors thank Dr. Dushyant Garg and Dr. Ruma Karmakar for continuous help in research experiments.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Authors and Affiliations

  1. 1.Kusuma School of Biological SciencesIndian Institute of Technology DelhiNew DelhiIndia

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