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Follistatin domain containing proteins

A technology of follicle-stimulating protein and protein, which is applied in the treatment of diseases related to metabolism and regulation, diseases and diseases related to adipose tissue and bone degeneration, and can solve problems such as unknown functions

Inactive Publication Date: 2007-07-04
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Other follistatin domain proteins have been described or published in the NCBI database (National Center for Biotechnology Information, Bethesda, MD, USA), however their functions are currently unknown

Method used

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  • Follistatin domain containing proteins
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1: Purification of JA16-bound beads

[0127] N-hydroxysuccinimide activated beads (4% agarose beads, Sigma H-8635, St Louis MO) in MilliQ-H 2 Washed in O and incubated with anti-GDF-8 JA16 monoclonal antibody (3-4 μg / μl in 100 mM MOPS, pH 7.5) at a ratio of 10 mg JA16 / ml final resin concentration for 4 hours at 4°C. Beads were washed extensively with 100 mM MOPS pH 7.5 and phosphate buffered saline (PBS) (Ausubel et al., (1999) Current Protocols in Molecular Biology, John Wiley and Sons) and stored in PBS at 4°C until use. Control beads were prepared in the same manner but without the JA16 antibody.

Embodiment 2

[0128] Example 2: Affinity purification

[0129] A total of 40 μl of packaged JA16-conjugated beads or control beads were incubated with 15 ml of normal Balb / C mouse serum (Golden West Biologicals, Temecula CA) or 30 ml of collected normal human serum (ICN Biomedical, Aurora OH) at 4°C. Incubate for 3 hours. Beads were washed twice in -10 ml cold 1% Triton X-100 / PBS, twice in -10 ml cold 0.1% Triton X-100 / PBS, and twice in -1 ml cold PBS. Proteins were eluted from the beads in 3 subsequent steps. First, the beads were subjected to a 'mock elution' treatment where 100 μl PBS was added to the beads and incubated at 4°C for 30 minutes. Supernatants were collected and combined with 30 μl 4×LDS sample buffer (Invitrogen, Carlsbad CA). Second, the beads are subjected to 'peptide elution' by adding 1 μg / μl of the competing peptide (sequence:

[0130] DFGLDSDEHSTERSSRYPLTVDFEAFGWD-COOH (SEQ ID NO: 12) was added to the beads and incubated for an additional 30 minutes at 4°C. Super...

Embodiment 3

[0132] Example 3: Mass spectrometry

[0133] Samples were reduced with NuPage 10X Reducing Agent (Invitrogen) for 10 minutes at 80°C and alkylated with 110 μΜ iodoacetamide for 30 minutes at 22°C in the dark. Samples were immediately run on a 10% NuPage Bis-Tris gel according to the manufacturer's recommendation (Invitrogen) in the MES buffer system and silver stained using a glutaraldehyde-free system (Shevchenko et al., (1996) Anal.Chem. , 68: 850-858). Bands were excised and digested with sequencing-grade modified trypsin (Promega, Madison WI) in Abimed DigestPro (Langenfeld, Germany) or ProGest Investigator (Genomics Solutions, Ann Arbor MI). Digested samples were reduced in volume by evaporation and made up to a final volume of -20 μl with 1% acetic acid. Samples (5-10 μl) were loaded onto a 10 cm x 75 μm id C18 reverse phase column packed in Picofrit needles (New Objectives, Woburn MA). MS / MS data were collected using a LCQ Deca or LCQ Deca XP (Finnigan, San Jose CA) ...

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Abstract

The present invention relates to the use of proteins comprising at least one follistatin domain to modulate the level or activity of growth and differentiation factor-8 (GDF-8). More particularly, the invention relates to the use of proteins comprising at least one follistatin domain, excluding follistatin itself, for treating disorders that are related to modulation of the level or activity of GDF-8. The invention is useful for treating muscular diseases and disorders, particularly those in which an increase in muscle tissue would be therapeutically beneficial. The invention is also useful for treating diseases and disorders related to metabolism, adipose tissue, and bone degeneration.

Description

[0001] This application claims the benefit of US Provisional Application No. 60 / 357,846, filed February 21, 2002, and US Provisional Application No. 60 / 434,645, filed December 20, 2002. field of invention [0002] The present invention relates to the use of proteins comprising at least one follistatin domain to modulate the level or activity of growth and differentiation factor-8 (GDF-8). More particularly, the present invention relates to the use of proteins comprising at least one follistatin domain, excluding follistatin itself, for the treatment of disorders associated with modulation of GDF-8 levels or activity. The present invention is useful in the treatment of muscle diseases and conditions, particularly those in which muscle tissue increases. The invention is also useful in the treatment of diseases and conditions associated with metabolism, adipose tissue and bone degeneration. Background of the invention [0003] Growth and differentiation factor-8 (GDF-8), also ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N37/18A61K38/16
Inventor J·J·希尔N·M·沃尔夫曼
Owner WYETH LLC
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