Authors:

Julia Rose Millikin

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About Paper:

Gq is a guanine nucleotide-binding protein (G protein) involved in signal transduction composed of three different protein chains. It is activated by G protein-coupled receptors (GPCRs) on the cell surface in response to external stimuli, releasing the bound G protein heterotrimers as the subunits Galphaq and Gbetagamma. Deactivation of the GPCR is initiated by a G protein receptor kinase (GRK), whose activity in pathophysiological conditions has been linked to heart disease and cancer. To continue structural studies on GRKs and GPCRs, a supply of active Galphaq is useful for binding to GRK2-GPCR complexes, increasing their stability and hopefully resolution in cryo-EM studies. Galphaq for structural studies is typically expressed in insect cells, which have high cost and time requirements. Utilizing E. coli cells will mitigate both time and cost. To optimize E. coli expression, Galphaq can be tagged to increase solubility, and post-induction temperature altered to reduce protein aggregation. To this end, hexahistidine and SUMO tags were added to the N-terminus and a FLAG tag added to the C-terminus. Two post-induction conditions were selected, 30 degrees Celsius for 12 hours and 20 degrees Celsius for 20 hours. Aliquots of Galphaq- transformed bacterial cells were grown to optimum density, and then expression was induced and temperatures altered. Anticipated results include active, properly folded Galphaq protein expressed in E. coli cells, detected by molecular weight and a trypsin protection assay. Using this optimized expression protocol, Galphaq can be produced for cryo-EM studies, increasing overall quality of GRK2-GPCR structures. Keywords: G Protein; GPCR; Protein Expression; E. coli

Source:

Purdue University / 2025

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Co-authors:

Julia Rose Millikin