Expression, Purification And Crystallization Of Drosophila GABA_B Receptors C-terminal Coiled-coil Domain Complex

Metabotropic GABAB receptors in the central nervous system are receptors of the main inhibitory neurotransmitter ? – aminobutyric acid, which are class C G-protein coupled receptors and located in the cell membrane. Their topological structure contains N-terminal ectodomain, transmembrane domain and C-terminal coiled-coil domian. Human GABAB receptors function as an obligatory heterodimer of GB1 and GB2 subunits, and GB1 is responsible for binding of ligands while GB2 is responsible for G-protein coupling to activate downstream signaling pathway. GB2 can express in cell membrane alone, but GB1 cannot since the C-terminal endoplasmic reticulun(ER) retention signal prevent its expression in cell membrane alone. However, when both GB1 and GB2 are express together, their C-terminal can strongly interact with each other as a coiled-coil domain complex which can shield the function of ER retention so that these two subunits exist as heterodimer.Unlike in Human, Drosophila GABAB receptors contain DGB1, DGB2 and DGB3 subunits which the former two can also assemble functional heterodimer while the physical function of DGB3 is still unknown. In this report, focusing on C-terminal amino acid suquence, first I did protein secondary structure prediction by bioinformatic ways and found out the probable ?-helix area. Then, refering to published crytal structure of Human GABAB receptors C-terminal coiled-coil domain complex, I designed six groups of amino acid sequence after sequence alignment of Human and Drosophila GABAB receptors. All of these sequence were constructed to vector of His- or GST-tag to express and purify these recombinant protein in E. Coli expression system. These purified recombinant protein were then used in GST – Pull Down assay to check whether there is strong interaction and found the amino acid sequence area of such a kind of interaction. Finaly, I designed a group of DGB1 and DGB2 C-terminal amino acid and got these recombinant protein after my explored best ways of expression and purification which were then used in screening of crystal growth conditions.This report is important for structural research of Drosophila GABAB receptors C-terminal, expected to help explain the expression mechanism in cell membrane of such functional heterodimer and lays foundations for further research on sturcture and function of GABAB receptors.