Abstract
The interaction between the synaptic adhesion molecules neuroligins and neurexins is essential for connecting the pre- and post-synaptic neurons, modulating neuronal signal transmission, and facilitating neuronal axogenesis. In chapter 3, we describe the simultaneous expression of the extracellular domain of neuroligin 1 (NL1) proteins along with the enhanced green fluorescent protein (EGFP) using the bi-cistronic baculovirus expression vector system (bi-BEVS). Recombinant NL1 proteins were secreted into the culture medium and the optimum harvest time for NL1 proteins based on the lysis of infected cells was determined by the release of cytosolic EGFP. The NL1 protein (129 ± 13 μg / 8x107 High Five cells; ~96% purity by metal affinity chromatography) was obtained from the supernatant of the recombinant virus-infected insect cells. A novel chip was employed to address whether the recombinant NL1 is functional in axogenesis. The purified NL1 promoted and enhanced the growth rate (137.07 ± 9.74 μm/day) of the axon on NL1/PLL (poly-L-lysine)-coated fine lines on the chip compared to those lines that were coated with PLL alone (105.53 ± 4.53 μm/day). These results were confirmed by fluorescence immunocytochemistry and demonstrated that the recombinant protein can be purified by a one-step process using IMAC combined with monitoring of cell lysis by bi-BEVS. This technique along with our novel chip offers a simple, cost-effective and useful platform for understanding the roles of NL1 protein in neuronal regeneration and synaptic formation studies. NMDA receptors are critical mediators of excitatory neurotransmission in the brain, being pivotal for long term potentiation. NMDA receptor channels are highly permeable to calcium ions, and thus overactivation leads to excitotoxic neuronal cell death. Functional NMDA receptors are formed from the co-assembly of the obligatory NR1 glycine-binding subunit with NR2 and/or NR3 subunits. However, the quaternary structure of NMDA receptors is still not yet established. To functional express these important heteromeric NMDA receptors, a polycistronic eucaryotic expression system is required. In chapter 4, we had constructed a tri-cistronic expression votor by the inclusion of two internal entry sites (IRESs) and demonstrated that three genes can be co-expressed by this technique. In chapter 5, we employed the novel baculovirus expression vectors to express NMDA receptors in Sf21 insect cells. Western blot and immunostaining demonstrated the expression of NMDA receptors on plasma membrane. Calcium image analysis indicated the function of this recombinant NMDA is responsible to NMDA and glutamate. These studies will facilitate the protein structure determination of NMDA receptor.