The rat serotonin reuptake transporter (rSERT) is a sodium-chloride-dependent transporter that reuptakes serotonin back into the presynaptic neurons after delivering its chemical message. Serious mental health illnesses are possible when SERT is not functioning normally, therefore a complete structure of the transporter and better understanding of its allostery is necessary to develop targeted drugs and therapies that effectively treat these disorders. Due to the large size of SERT and the fluidity of the extracellular loops, which play an important role in transportation, this information is difficult to obtain by traditional structural methods. As a complementary alternate approach, crosslinking studies will be performed to determine nearby structure and interactions of the extracellular loops in which a single reactive thiol was introduced at selected sites. The work presented will be reagrding bacmid preparation, viral amplification, and protein purification performed on rSERT X8C constructs in preparation for further crosslinking studies. L406C rSERT was made by PCR mutagenesis, the transfer plasmid was used to produce recombinant bacmid which was then used to transfect SF9 cells for baculovirus production. The baculovirus was amplified and the rSERT protein was harvested from infected insect cells and purified. Future studies will involve heterobifunctional crosslinking via disulfide attachment at the site of thiol mutation with MTS-benzophenone. Upon photoactivation, subsequent determination of sites of crosslinking in rSERT peptide fragments via tandem mass spectrometry to determine residues in a roughly 20Å radius of location 406C in the rSERT protein stabilized in the open conformation.

Baculovirus expression, amplification, and protein purification of recombinant rat serotonin transporter containing a single point-mutation for future crosslinking studies

253rd National Meeting (2017)

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