Dana-Farber and Boston Children’s Cancer and Blood Disorders Center Research Update

Thanks to the TeamConnor funding, I have been able to purchase many reagents to support my research related to miniprotein binders of PAX3-FOXO1 oncogenic fusion protein. I have purchased E. Coli expression constructs and have purified the second domain of PAX3, the homeobox domain. I have characterized that this protein is folded in an alpha-helical configuration by CD spectroscopy. I am about to embark on screening via yeast display libraries for binders to this portion of the PAX-FOXO1 fusion protein. The FOXO1 protein has been harder to purify from E. Coli, so I am now attempting to optimize expression within mammalian cells (Expi293s) as well as SF9 cells. TeamConnor support has allowed me to purchase the reagents for the mammalian cell approaches. Once purified, I will screen binders for this component of the oncogenic fusion. 

In parallel, I have begun in vitro screening of miniprotein scaffolds to ensure there is no off-target toxicity of the base scaffolds in alveolar rhabdomyosarcoma cell lines. I have obtained both normal skin fibroblasts, embryonal as well as alveolar rhabdomyosarcoma cell lines to use for purposes of screening the miniprotein scaffolds. Once the binders are isolated from yeast display screening, I will then introduce the specific PAX3 and FOXO1 binders and screen for binding in vitro.

Researcher:  Alyssa Kennedy, MD, PhD

Dana-Farber and Boston Children’s Cancer and Blood Disorders Center