Synaptogenic protein tagged with biotin and reconstitution of artificial synapse by using same

Abstract

Disclosed is a polypeptide containing an extracellular domain of a synaptogenic protein, and a method for manufacturing a nerve cell, a complex containing a biotin tagged at the C-terminus of the polypeptide, an artificial synapse inducer for coupling the composite to a streptavidin (SAV)-coated substrate and a nerve cell. The complex tagged with a biotin at the C-terminus of the polypeptide containing the extracellular domain of the synaptogenic protein, such as neuroligin-1, can display activity by being attached to the SAV-coated substrate to adjust the orientation thereof without help of a supported lipid bilayer. The complex containing an additional RFP between the extracellular domain and the biotin of the synaptogenic protein not only facilitates easier mass-production, quantification, and tracking, but also displays activity of a normal synaptogenic protein, thereby inducing excitatory or inhibitory synaptic differentiation by being fixed to the substrate and added to the nerve cell culture.

1 . A complex comprising a polypeptide comprising an extracellular domain of a synaptogenic protein; and biotin tag at the C-terminus of the polypeptide. 
     
     
         2 . The complex of  claim 1 , wherein the synaptogenic protein is selected from the group consisting of neuroligin, neurexin, leucine-rich repeat transmembrane protein (LRRTM), netrin G ligand (NGL), synaptic cell adhesion molecule (SynCAM), ephrin-B receptor (EphB), leukocyte common antigen-related protein (LAR), netrin G, and Slitrk (Slit- and Trk-like proteins). 
     
     
         3 . The complex of  claim 1 , wherein the synaptogenic protein is neuroligin-1. 
     
     
         4 . The complex of  claim 1 , further comprising a fluorescent protein in the order of the polypeptide comprising an extracellular domain of a synaptogenic protein, the fluorescent protein, and the biotin from a N-terminus thereof. 
     
     
         5 . The complex of  claim 4 , wherein the fluorescent protein is red fluorescent protein (RFP). 
     
     
         6 . The complex of  claim 1 , further comprising a polyhistidine-tag (His-tag) or influenza hemagglutinin epitope tag (HA-tag). 
     
     
         7 . An artificial synapse inducer, wherein the complex of claim  1 is attached to a substrate coated with a biotin-binding protein. 
     
     
         8 . The artificial synapse inducer of  claim 7 , wherein the biotin-binding protein is selected from the group of avidin-like proteins consisting of streptavidin, traptavidin, and neutravidin. 
     
     
         9 . The artificial synapse inducer of  claim 7 , wherein the substrate is a microbead. 
     
     
         10 . A method for preparing a presynaptic differentiation-induced neuron comprising culturing the neuron in a medium comprising the artificial synapse inducer of  claim 7 . 
     
     
         11 . The method of  claim 10 , wherein the presynaptic differentiation is an excitatory presynaptic differentiation or an inhibitory presynaptic differentiation. 
     
     
         12 . The artificial synapse inducer of  claim 7 , wherein the synaptogenic protein is selected from the group consisting of neuroligin, neurexin, leucine-rich repeat transmembrane protein (LRRTM), netrin G ligand (NGL), synaptic cell adhesion molecule (SynCAM), ephrin-B receptor (EphB), leukocyte common antigen-related protein (LAR), netrin G, and Slitrk (Slit- and Trk-like proteins). 
     
     
         13 . The artificial synapse inducer of  claim 7 , wherein the synaptogenic protein is neuroligin-1. 
     
     
         14 . The artificial synapse inducer of  claim 7 , wherein the complex further comprises a fluorescent protein in the order of the polypeptide comprising an extracellular domain of a synaptogenic protein, the fluorescent protein, and the biotin from a N-terminus thereof. 
     
     
         15 . The artificial synapse inducer of  claim 14 , wherein the fluorescent protein is red fluorescent protein (RFP). 
     
     
         16 . The artificial synapse inducer of  claim 7 , wherein the complex further comprises a polyhistidine-tag (His-tag) or influenza hemagglutinin epitope tag (HA-tag).