Expression and purification of p300 CH1 and HIF-1α CTAD complex and screening of protein-protein interaction inhibitors

다국어 초록 (Multilingual Abstract)

The p300 CH1/HIF-1α CTAD interaction plays a key role in the transcription of hypoxia inducible genes and the inhibition of the protein-protein interaction can be a target for the development of cancer therapeutics since HIF-1 allows tumor growth in hypoxic environments by promoting angiogenesis and metabolic adaptations to hypoxia. Both proteins were expressed in E. coli and purified. By using NMR spectroscopy, the conformational status of p300 CH1/HIF-1α CTAD complex was confirmed. The p300 CH1 and HIF-1α CTAD were unstructured or partially structured without their binding partners; however, their complex had well-defined folded structures. The p300 CH1 and HIF-1α CTAD were co-purified and subjected to the measurement of 1H-15N HSQC spectrum. The fragment compounds were titrated to the complex and 1H-15N HSQC spectra were measured. Several compounds showed significant spectra change upon the fragment titration. Thus, based on the NMR measurement inhibitors of p300 CH1/HIF-1α CTAD interaction can be screened and the selected fragment compounds can be utilized to generate the lead molecules for cancer therapeutics.

목차 (Table of Contents)

• Abstract i
• ACKNOWLEDGEMENTS iv
• LIST OF FIGURES v
• LIST OF TABLE vi
• List of abbreviations vii
• Introduction 1
• Hypoxia-inducible factor-1 (HIF-1) CTAD and p300 CH1 protein 1
• Heteronuclear Single Quantum Correlation (HSQC) - 1H-15N HSQC in Protein NMR 3
• METERIALS AND METHODS 7
• Cloning 7
• Protein expression and purification 8
• NMR spectroscopy 11
• NMR titration fragment compounds 12
• RESULTS 13
• Cloning p300 CH1 and HIF-1α CTAD into pET21a and pGST vector 13
• Expression and purification of p300 CH1 and HIF-1α CTAD complex 14
• NMR spectra of p300 CH1 domain and HIF-1α CTAD complex 20
• Fragment screening 23
• Screening and discovery of protein-protein interaction inhibitors 25
• DISCUSSION 26
• References 28