title: Functional expression of rat neuroligin-1 extracellular fragment by a bi-cistronic baculovirus expression vector.
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. Here, we describe the simultaneous expression of the extracellular domain of rat neuroligin-1 (NL1) proteins along with the enhanced green fluorescent protein (EGFP) using the bi-cistronic baculovirus expression vector system (bi-BEVS). Recombinant rat NL1 protein, fused with signal sequence derived from human Azurocidin gene (AzSP), was secreted into the culture medium and the optimum harvest time for NL1 protein before the lysis of infected cells was determined through the release of cytosolic EGFP. The NL1 protein (0.129±0.013 mg/8×10(7) 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 rat 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.
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Wen-Shuo
authors.initials: WS
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Chung Yuan Christian University, Chungli 320, Taiwan.
authors.forename: Oliver B
authors.initials: OB
authors.lastname: Villaflores
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Co-Fan
authors.initials: CF
authors.lastname: Lu
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Huei-Ing
authors.initials: HI
authors.lastname: Wu
authors.affiliation: Department of Bioscience Technology, Chung Yuan Christian University, Chungli 320, Taiwan.
authors.forename: Yu-Jei
authors.initials: YJ
authors.lastname: Chen
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Chao-Yi
authors.initials: CY
authors.lastname: Teng
authors.affiliation: Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Yen-Chung
authors.initials: YC
authors.lastname: Chang
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Shou-Lin
authors.initials: SL
authors.lastname: Chang
authors.affiliation: Department of Bioscience Technology, Chung Yuan Christian University, Chungli 320, Taiwan; R&D Center of Membrane Technology, Chung Yuan Christian University, Chungli 320, Taiwan. Electronic address: tywu@cycu.edu.tw.
authors.forename: Tzong-Yuan
authors.initials: TY
authors.lastname: Wu
mesh_terms: D000818:Animals; D001369:Axons / Q000187:drug effects; D016367:Baculoviridae / Q000235:genetics*; D015816:Cell Adhesion Molecules, Neuronal / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D009036:Moths; D009474:Neurons; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C093438:neuroligin 1; D049452:Green Fluorescent Proteins