Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof

a technology of functional conformational state and protein binding domain, which is applied in the field of gpcr structure biology and signaling, can solve the problems of biochemical instability and difficulty in obtaining high-resolution crystal structure, incompatible structural heterogeneity with crystal formation, and difficulty in obtaining active state of gpcr structur

Inactive Publication Date: 2013-05-30
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
View PDF1 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018]A third aspect hereof relates to a cellular composition comprising a protein binding domain hereof and/or a complex hereof. Preferably, the protein binding domain

Problems solved by technology

While this structural plasticity and dynamic behavior is essential for normal function, it contributes to their biochemical instability and difficulty in obtaining high-resolution crystal structures.
Obtaining structures of an active state of a GPCR

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof
  • Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof
  • Protein binding domains stabilizing functional conformational states of gpcrs and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Immunization, Library Construction and Initial Screening

[0275]To obtain in vivo matured nanobodies against β2AR, a llama (Llama glama) was immunized with recombinant β2AR truncated at Gly365 (β2AR-365) to exclude an immune response to the carboxyl terminus. β2AR-365 was expressed in insect cells and antigen was reconstituted as previously described (Day et al. 2007). After six weekly administrations of the reconstituted truncated agonist-bound receptor, lymphocytes were isolated from the blood of the immunized llama and a phage library prepared and screened as described in Materials and Methods to the Examples (see further). Two screens identified conformational nanobodies that recognize the native β2AR, but not the denaturated receptor.

example 2

Selection of Conformational-Specific Nanobodies by ELISA

[0276]In a first screen we compared the binding of the nanobodies on the native and heat denatured β2AR antigen in an ELISA. For each nanobody, one well was coated with phospholipid vesicles containing agonist-bound β2AR-365 (0.1 μg protein / well). Next, this plate was incubated at 80° C. for two hours. Next, another well of the same plate was coated with phospholipid vesicles containing agonist-bound β2AR-365 (0.1 μg protein / well) without heating. All of the nanobodies were able to selectively bind the native receptor but not the heat inactivated receptor, indicating that 16 binders recognize conformational epitopes.

example 3

Selection of Conformational-Specific Nanobodies by Dot Blot

[0277]In a next screen we compared the specificity of the nanobodies for a native agonist-bound β2AR receptor, versus a native inverse agonist-bound receptor, versus an SDS denaturated receptor by dot blot analysis. The screen identified 16 different conformational nanobodies that recognize native agonist-bound β2AR-365, but not the inverse agonist, or the heat denatured receptor (FIG. 2 (dot blots)).

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Temperatureaaaaaaaaaa
Molar densityaaaaaaaaaa
Molar densityaaaaaaaaaa
Login to view more

Abstract

The present invention relates to the field of GPCR structure biology and signaling. In particular, the present invention relates to protein binding domains directed against or capable of specifically binding to a functional conformational state of a G-protein-coupled receptor (GPCR). More specifically, the present invention provides protein binding domains that are capable of increasing the stability of a functional conformational state of a GPCR, in particular, increasing the stability of a GPCR in its active conformational state. The protein binding domains of the present invention can be used as a tool for the structural and functional characterization of G-protein-coupled receptors bound to various natural and synthetic ligands, as well as for screening and drug discovery efforts targeting GPCRs. Moreover, the invention also encompasses the diagnostic, prognostic and therapeutic usefulness of these protein binding domains for GPCR-related diseases.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a national phase entry under 35 U.S.C. §371 of PCT International Patent Application PCT / EP2011 / 062288, filed Jul. 18, 2011, designating the United States of America and published in English as International Patent Publication WO 2012 / 007593 A1 on Jan. 19, 2012, which claims the benefit under Article 8 of the Patent Cooperation Treaty and under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 61 / 399,781, filed Jul. 16, 2010, and under Article 8 of the Patent Cooperation Treaty to Great Britain Patent Application Serial No. 1014715.5, filed Sep. 6, 2010.GOVERNMENT RIGHTS[0002]This invention was made with Government support under Contract No. NS028471 awarded by the National Institutes of Health. The Government has certain rights in this invention.STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)—SEQUENCE LISTING SUBMITTED AS A TXT AND PDF FILES[0003]Pursuant to 37 C.F.R. §1.821(c) or (e), files containin...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): G01N33/68
CPCA61K38/00C07K14/705C07K14/723G01N33/566G01N2333/726C07K2317/75C07K16/28C07K2317/22C07K2317/33C07K2317/569G01N33/68A61P25/00A61P31/04A61P35/00A61P37/06A61P9/00C07K2317/51C07K2317/56G01N23/20G01N33/6857G01N33/6872
Inventor STEYAERT, JANPARDON, ELSLAEREMANS, TOONRASMUSSENKOBILKA, BRIANFUNG, JUAN
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap