Innovative discovery of therapeutic, diagnostic and antibody compositions related to protein fragments of glutaminyl-tRNA synthetase
A therapeutic composition, technology of synthesizing enzymes, applied in the field of polynucleotides and their complements
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 217
[0074] I. Overview
[0075] The present invention relates at least in part to the discovery of novel AARS polypeptides, and methods for their preparation and use, which represent the conversion of a native wild-type protein into a new species exhibiting significantly different properties compared to the naturally occurring full-length glutaminyl tRNA synthetase gene. form. The identification of such AARS polypeptides was based on extensive sequence and mass spectrometric analysis of glutaminyl tRNA synthetases expressed in different tissues, followed by systematic generation and testing of each potential AARS polypeptide to identify those representing stable and soluble protein domains. Protein sequences that exhibit novel biological activities and favorable therapeutic drug properties.
[0076] Based on this analysis, several new families of AARS polypeptides derived from glutaminyl tRNA synthetases have been identified.
[0077] In one aspect, such glutaminyl tRNA syntheta...
Embodiment 1
[0772] Identification of proteolytic fragments and alternative splicing products from AARS using a protein topology and migration analysis platform
[0773] To identify AARS fragments from cell lines, conditioned media, and tissues, prepare samples as follows:
[0774] Mouse macrophages (RAW 264.7), cytosol and conditioned media: Use 15x 10 in serum-free DMEM 6 Density of cells / flask Treat cells. After 48 hours, conditioned media and cell pellets were collected and processed. 200 μg of protein were separated by SDS-PAGE from secretory and cytosolic proteome fractions, and gel slices were prepared for analysis by mass spectrometry.
[0775] Mouse pancreas tissue: Pancreas were dissected from three mice, dounce homogenized, and sonicated in PBS containing protease inhibitors. The cytosolic proteome was separated by centrifugation and 200 μg of protein was separated by SDS-PAGE and gel slices were prepared for analysis by mass spectrometry.
[0776] Mouse liver tissue: livers...
Embodiment 2
[0781] Identifying splice variants using deep sequencing
[0782] Identification of splice variants of aminoacyl tRNA synthetases using high-throughput sequencing of cDNA libraries enriched for aminoacyl tRNA synthetase transcripts. Prepare cDNA templates from total RNA extracts of tissues such as adult and fetal brain and enrich for aminoacyl-tRNA synthesis using primer sequences specific for all annotated exons of all annotated human aminoacyl-tRNA synthetases and their associated proteins Enzyme transcripts.
[0783] Human total RNA was obtained from Clontech. For cell lines and mouse tissue samples, total RNA was extracted using the RNA Extraction II Kit (MN). Genomic DNA in total RNA samples was digested by DNase I. To obtain mature messenger RNA (mRNA), RNA samples were enriched twice by incorporation of poly-A+ RNA and by digestion of 5'-capped RNA by a 5'-phosphate-dependent exonuclease. Complementary DNA (cDNA) is synthesized from mature RNA using primers that ann...
PUM
| Property | Measurement | Unit |
|---|---|---|
| solubility (mass) | aaaaa | aaaaa |
| particle size | aaaaa | aaaaa |
| molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More