Optimization of expression and purification of triple-negative breast cancer biomarkers in Saccharomyces cerevisiae

• Kaia Griffin, Biological Sciences, University of Delaware

• Karl Schmitz, Biological Sciences, University of Delaware

Approximately 15% of breast cancer cases are classified as triple-negative breast cancer (TNBC). Patients with TNBC lack the three most common biomarkers used when making breast cancer diagnoses: hormone receptors for estrogen and progesterone, and little to no HER2 protein. Without these biomarkers, TNBC patients often go undiagnosed or misdiagnosed for longer periods of time. Additionally, ~80% of FDA-approved breast cancer therapeutics target the three biomarkers that TNBC lacks. Consequently, patients are often treated with chemotherapy rather than targeted therapies, resulting in lower survival rates for TNBC than for other forms of breast cancer. 

The cell-surface proteins mesothelin (MSLN) and epidermal growth factor receptor (EGFR) are frequently overexpressed in TNBC tumors. MSLN and EGFR are linked to exacerbated cell growth and tumor progression, highlighting them as candidates for new biomarkers or drug targets for TNBC. Both proteins require post-translational modifications (PTMs) for protein stability, notably glycosylation. Glycosylation is a critical sugar modification that promotes proper protein folding in vivo.  Bacterial cells are not capable of installing these PTMs, so we instead turned to eukaryotic cells to purify these proteins, ensuring they fold in active, stable forms. We cloned several plasmids encoding MSLN or domain III of EGFR (EGFRdIII) with alternative signal peptides to optimize protein expression in Saccharomyces cerevisiae. Specifically,  glycosylation of these proteins could impact subsequent in vitro assays, including crystallization. To combat this, we also expressed and purified Endoglycosidase H (EndoH), a deglycosylating enzyme from Streptomyces plicatus.  

The optimized S. cerevisiae expression and purification protocols developed here for MSLN and EGFRdIII will yield protein for downstream structure-based studies and for use in nanobody surface display to guide future biomarker testing or targeted drug development against TNBC.