Artificial synapse inducer and method of making the same

Patent metadata

Field	Value
Publication number	US-2019119343-A1
Application number	US-201816155209-A
Country	US
Kind code	A1
Filing date	Oct 9, 2018
Priority date	Apr 24, 2013
Publication date	Apr 25, 2019
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 . An artificial synapse inducer comprising:
a complex comprising a polypeptide and a biotin conjugated to the polypeptide, wherein the polypeptide comprises the sequence of SEQ ID NO: 11; and a substrate coated with a biotin-binding protein, wherein the biotin and the biotin-binding protein are bound together such that the complex is attached to the substrate without a lipid bilayer.

2 . The artificial synapse inducer of claim 1 , wherein the biotin-binding protein is selected from the group of avidin-like proteins consisting of streptavidin, traptavidin, and neutravidin.

3 . The artificial synapse inducer of claim 1 , wherein the substrate comprises a microbead.

4 . The artificial synapse inducer of claim 1 , the complex comprises a fluorescent protein.

5 . The artificial synapse inducer of claim 4 , wherein the fluorescent protein is selected from the group consisting of red fluorescent protein (RFP), blue fluorescent protein (BFP), green fluorescent protein (GFP) and yellow fluorescent protein (YFP).

6 . The artificial synapse inducer of claim 1 , wherein the complex further comprises a polyhistidine-tag (His-tag) or influenza hemagglutinin epitope tag (HA-tag).

7 . The artificial synapse inducer of claim 1 , wherein the biotin is conjugated to the polypeptide via a linking peptide connected to Isoleucine located at the C-terminus of SEQ ID NO: 11.

8 . The artificial synapse inducer of claim 7 , wherein the linking peptide comprises a glycine-serine linker (GS) and a biotin acceptor peptide (AP).

9 . A method for preparing a presynaptic differentiation-induced neuron, the method comprising:
culturing the neuron in a medium comprising the artificial synapse inducer of claim 1 .

10 . The method of claim 9 , wherein the presynaptic differentiation is an excitatory presynaptic differentiation or an inhibitory presynaptic differentiation.

11 . A method of forming the artificial synapse inducer of claim 1 , the method comprising:
providing a substrate coated with a biotin-binding peptide; providing a complex comprising a polypeptide and a biotin conjugated to the polypeptide, wherein the polypeptide comprises the sequence of SEQ ID NO: 11; and applying the complex onto the substrate for causing the biotin and the biotin-binding peptide to bind together such that the complex is attached to the substrate without a lipid bilayer, thereby forming the artificial synapse inducer.

12 . The method of claim 11 , wherein the biotin is conjugated to the polypeptide via a linking peptide connected to Isoleucine located at the C-terminus of SEQ ID NO: 11.

13 . The method of claim 12 , the linking peptide comprises a glycine-serine linker (GS) and a biotin acceptor peptide (AP).

14 . The method of claim 11 , wherein the artificial synapse inducer further comprises a fluorescent protein introduced into the complex.

15 . The method of claim 11 , wherein the fluorescent protein comprises red fluorescent protein (RFP), blue fluorescent protein (BFP), green fluorescent protein (GFP) and yellow fluorescent protein (YFP).

16 . The method of claim 11 , wherein the complex further comprises a polyhistidine-tag (His-tag) or influenza hemagglutinin epitope tag (HA-tag).

Assignees

• Seoul Nat Univ R&Db Foundation

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

• C07K2319/90

  containing a motif for post-translational modification · CPC title

• C07K2319/42

  containing a HA(hemagglutinin)-tag · CPC title

• C07K2319/21

  containing a His-tag · CPC title

• C07K2319/40

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