Protein Expression and Purification Combined with FPOP to Identify GCaMP2 Structure

Graduate #38
Discipline: Chemistry and Chemical Sciences
Subcategory: Chemistry (not Biochemistry)

Cord Carter - Jackson State University
Co-Author(s): Emily Hart, Maissa Gaye, Lisa Jones, Indiana University Purdue University Indianapolis, Indianapolis, IN

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The work presented here is focused on the characterization of the fusion protein, GCaMP2. This protein was cloned into the pRSET bacterial expression vector for expression in Escherichia coli. In recent years, protein fusion tags have been used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Immobilized metal-affinity chromatography (IMAC) has been used to purify recombinant proteins that have a polyHistidine (polyHis) tag. Histidine exhibits the strongest interaction with immobilized metal ion matrices due to its electron donor group on the imidazole ring group. We will structurally analyze the purified GCaMP2 using the MS-based protein footprinting method fast photochemical oxidation of proteins (FPOP), which utilizes hydroxyl radicals to oxidatively modify solvent accessible residues of proteins. The hydroxyl radicals are generated by the photolysis of hydrogen peroxide by a pulsed excimer laser. There is a crystal structure available for GCaMP2 which can be used as a model system to test how well FPOP works. For this study, we want to use the FPOP to map the structural differences between calcium-bound and calcium-free form of GCaMP2. We will use EGTA as a chelating agent for the calcium-free form because it has a higher affinity for the calcium. In order to do FPOP on the protein we have to get the protein. So we are expressing in E.coli and purifying it using polyHis tag. We were able to visualize a green distinctive color when the GCaMP2 was purified. We are now able to test how well FPOP works on the protein.

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Funder Acknowledgement(s): Indiana University Historically Black Colleges & Universities STEM Initiative, Jackson State University Louis Stokes Mississippi Alliance for Minority Participation Bridge to the Doctorate

Faculty Advisor: Lisa Jones, joneslis@iupui.edu