Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Methods and compositions using KLOTHO-FGF fusion polypeptides

A technology of fusion polypeptide and composition, applied in the field of treating testicular pathology, fusion polypeptide, Klotho fusion polypeptide

Inactive Publication Date: 2011-04-13
NOVARTIS AG
View PDF32 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is speculated that tissue-specific expression of α-Klotho confines the activation of FGF23 signaling to these tissues

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
  • Methods and compositions using KLOTHO-FGF fusion polypeptides
  • Methods and compositions using KLOTHO-FGF fusion polypeptides
  • Methods and compositions using KLOTHO-FGF fusion polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0208] Expression and purification of embodiment 1.Klotho fusion polypeptide

[0209] Expression of Klotho fusion polypeptide

[0210] The polypeptide of the present invention is prepared by transiently transfecting HEK293T cells with an expression vector encoding a Klotho fusion polypeptide having the extracellular domain of αKlotho and the FGF23(R179Q) variant. Conditioned media containing the expressed polypeptides were generated by transient transfection of the corresponding expression plasmids for Klotho, FGF23 and Klotho-FGF23(R179Q) fusion proteins. Transfections were performed in 6-well plates using Lipofectamine 2000 (Invitrogen, catalog #11668-019). Five hours after transfection, the transfection mixture was replaced with 3 ml DMEM plus 1% FBS. Conditioned medium was collected 72 hours after the addition of 3 ml DMEM plus 1% FBS. Conditioned medium samples from various transiently transfected HEK293T cells were separated by SDS-polyacrylamide gel electrophoresis (...

Embodiment 2

[0214] Example 2. In vitro assays to assess the activity of Klotho fusion polypeptides

[0215] Egr-1-luciferase

[0216] The expressed αKlotho fusion polypeptides were tested for biological activity in an Egr-1-luciferase reporter assay. Binding of the Klotho fusion polypeptide to the FGF23 receptor results in downstream activation of Egr-1 and expression of a luciferase reporter regulated by the Egr-1 promoter. The Egr-1-luciferase reporter gene was constructed based on the report of Urakawa et al. (Nature, 2006, Vol. 444, 770-774). HEK293T cells seeded in 48-well poly-D-lysine plates were transfected with the Egr-1-luciferase reporter gene together with the transfection labeling reporter gene (renilla luciferase). Five hours after Egr-1 luciferase reporter transfection, the transfection mixture was replaced with 3 ml DMEM plus 1% FBS. Conditioned medium was collected 72 hours after the addition of 3 ml DMEM plus 1% FBS. After 5 hours, the transfection mixture was replac...

Embodiment 3

[0222]Example 3. In vitro assays to assess the effects of Klotho fusion polypeptides on muscle cells.

[0223] The biological effects of the expressed Klotho fusion polypeptides were tested on C2C12 myoblasts. Treatment of C2C12 myoblasts with IGF-1, FGF2 or sKlotho-FGF23 results in myotube growth and phosphorylation of signaling proteins. C2C12 myoblasts were seeded at a density of 40,000 cells / well on 6-well poly-D-lysine and fibronectin-coated plates in growth medium [(3 parts DMEM and 1 part F12), 10% FBS, 1 % Glut; 1% P / S; 1% Linoleic Acid; 0.1% ITS: [Insulin (10 mg / ml), Transferrin (5.5 mg / ml) and Selenium (5 ng / ml)]. After myoblasts reached confluence (3 days), the medium was changed to differentiation medium (DMED with 2% horse serum; 1% Glut; 1% P / S).

[0224] For myotube diameter experiments, 3 days after confluence, the medium was changed to differentiation medium in which IGF-1 (10 nM), FGF2 (20 ng / ml) or sKlotho-FGF23 (20nM) to treat the cells for 24 hours. A...

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

No PUM Login to View More

Abstract

The present invention is directed to methods, kits and compositions for preventing or treating age-related conditions or metabolic disorders. The Klotho fusion polypeptides of the invention include at least a Klotho protein or an active fragment thereof. The Klotho fusion proteins are useful in the treatment and preventionofa variety of age-related conditions and metabolic disorders.

Description

1. Background technology [0001] The α-Klotho gene encodes a 130 kDa single-pass type I transmembrane protein with an extracellular domain and a short cytoplasmic domain. The extracellular domain of α-Klotho protein includes 2 subdomains called KL-D1 and KL-D2. These 2 subdomains share sequence homology with bacterial and plant β-glucosidases. The extracellular domain of α-Klotho protein can bind to the cell surface through the transmembrane domain, or can be cleaved and released into the extracellular environment. Fragmentation of the extracellular domain appears to be facilitated by low local extracellular Ca2+ concentration. [0002] In addition to α-Klotho, a homologue of α-Klotho, β-Klotho, has been identified (Ito et al., Mech. Dev. 98:115-9 (2000)). β-Klotho is also a single-span type I transmembrane protein with extracellular KL-D1 and KL-D2 subdomains. [0003] Modulation of α-Klotho expression has been shown to cause senescence-associated features in mammals. Mic...

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): C07K14/50C12N15/62A61K38/18
CPCC12N9/2402C07K2319/00A61K38/00C07K14/435C07K14/50C12Y302/01031A61P11/00A61P13/00A61P13/08A61P13/12A61P17/16A61P19/02A61P19/10A61P21/00A61P21/02A61P25/14A61P25/28A61P27/12A61P27/16A61P3/00A61P3/04A61P3/12A61P35/00A61P37/02A61P43/00A61P9/10A61P9/12A61P3/10
Inventor D·格拉斯S-I·胡
Owner NOVARTIS AG
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com