Characterizing Novel Kinases as an Authentic Research Experience in the Undergraduate Biochemistry Lab

Abstract

The protein databank (PDB) contains 183,000 structures, with approximately 4200 of those currently classified as having “unknown function”. Based on structures with known function(s) in the PDB, about 2500 of those proteins are expected to be enzymes. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) consortium has developed a series of modules that guide undergraduate students through in silicoprediction of the function of proteins of “unknown” function, along with modules for in vitro confirmation of these predictions. We used these modules as part of a kinase‐focused course‐based undergraduate research experience (CURE). Kinases were selected because of their abundance in humans (over 500 members of the kinase superfamily) and their involvement in almost every aspect of cellular function. Multiple putative kinases were characterized by undergraduates, using the in silicoBASIL modules (which utilize BLASTp, Pfam, SPRITE and Dali). Specific potential substrates were then identified using SwissDock and/or AutoDock Vina (via PyRx). Students were able to successfully overexpress the putative kinases and subsequently purify them using affinity chromatography. Coupled in vitro kinase assays were performed first using the well‐characterized Abl kinase and an appropriate peptide substrate. Students then adapted the coupled kinase assay to evaluate the potential substrates they had identified. Students reported that having an integrated laboratory experience, with the ability to make their own decisions based on their in silicodata, gave them a strong sense of ownership of their projects. Additionally, the techniques they learned were all used to support their hypotheses, which helped them understand the details of why and how each method was performed. Overall, they described it as a positive experience and one of their more interesting laboratory courses.