Authors:

Carson Huber

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About Paper:

c-Myc is one of the most important oncogenes that is deregulated in about 80% of cancers. G-quadruplex is a globular DNA secondary structure. G-quadruplex formed in the c-Myc promoter region (MycG4) functions as a transcription silencer and is an attractive anticancer drug target. Nucleolin was discovered as a major MycG4 binding protein. It shows a remarkably high binding affinity for MycG4 and can repress the activity of the c-Myc promoter. Nucleolin's central RBD domain containing four RNA binding domains (NCL1234) is shown to be the high affinity minimal binding domain with MycG4. Fab is widely used to facilitate crystallization and we have discovered Fabs that specifically bind the NCL1234-MycG4 complex using a phage display screening. For my SURF research, I am working on the expression and purification of wildtype NCL1234 and its various mutants, as well as multiple Fab proteins. I expressed these proteins in E.coli bacteria cells. The NCL1234 proteins were purified by FPLC using Histrap affinity column, Q-seph ion-exchange column and size exclusion column. Fabs were purified by FPLC using protein L affinity column. I am able to obtain the proteins with large quantities and high purity, which are critical for structure study. I will use the purified NCL1234 and Fab proteins for NCL1234-MycG4-Fab ternary complex which will be used to set up crystallization trays for X-ray crystallographic structural study. Molecular level details of nucleolin protein recognition of MycG4 is important to understand MycG4 and nucleolin functions and help develop MycG4-targeted cancer therapeutics.

Source:

Purdue University / 2023

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Co-authors:

Carson Huber