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

Patent metadata

Field	Value
Publication number	US-2016137707-A1
Application number	US-201314786908-A
Country	US
Kind code	A1
Filing date	Aug 19, 2013
Priority date	Apr 24, 2013
Publication date	May 19, 2016
Grant date	—

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.

First claim

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).

Assignees

• Univ Seoul Nat R & Db Found

Inventors

• Chung Taek Dong
• Hwang In Seong
• Kim Eun Joong
• Jeon Chang Su

Classifications

• C07K14/705 Primary

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• C07K2319/40

  containing a tag for immunodetection, or an epitope for immunisation · CPC title

• C07K14/4756 Primary

  Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor · CPC title

• C07K2319/90

  containing a motif for post-translational modification · CPC title

• C12N2501/13

  Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins · CPC title