This document discusses the isolation and characterization of a novel protein, c19orf10, found in synovial tissue. During a proteomic analysis of fibroblast-like synoviocytes (FLSs) from rheumatoid arthritis (RA) and osteoarthritis (OA) patients, the uncharacterized protein c19orf10 was identified. The protein was purified using techniques like chromatography, precipitation, and SDS-PAGE. Its gene was located on chromosome 19 and was found to have multiple splice variants. Immunohistochemistry revealed that c19orf10 is produced by FLSs and synovial fibroblasts. While the role of c19orf10 is unknown, its presence in synovial fluid and differential expression in RA and

1. ISOLATION AND
CHARACTERIZATION
OF NOVEL PROTEIN
By
Shrushti Joshi
1850302

2. What are
novel
proteins
• The word "novel“ simply mean newly discovered or described.
It means dealing with a protein that's novel in the sense that it
hasn't been known previously.
• A protein that is new to science, or has a function that was
previously unknown.
• Estimates place the number of proteins in the human body at
approximately 21,000, many of which serve several functions
depending where in the body they are.
• So when a protein is described as novel, it means the protein in
question is being described for the first time, or has secondary
activity that was previously unknown.
Fig :- Discovery of the novel green fluorescent protein by NSRRC
2018/04/03

3. Protein
isolation
techniques
• Protein extraction from cell:-
1. Physical –Homogenizer, French press, sonication.
2. Biological – enymes like lysosomes, lipases etc
3. Chemical – lysis buffer, cell disruption compounds
like SDS.
• Purification of proteins
1. Chromatography
2. Evaporation
3. Precipitation
4. Salting out
5. SDS page
6. Immunoblotting

4. Protein
characterization
techniques
• Techniques include
1. SDS page
2. Western blotting
3. Biochemical assays on NATIVE page
4. Mass spectrophotometer
5. cDNA constructs and hybridization

5. The
identification
and
characterization
of a novel
protein,
c19orf10, in the
synovium
• The healthy synovial membrane consists of a thin layer of
fibroblast-like synoviocytes (FLSs) and macrophages.
• These cells produce glycosaminoglycans such as
hyaluronic acid and lubricating glycoproteins for secretion
into the synovial fluid.
• Healthy homeostasis within the joint can be disturbed by
development of inflammatory diseases such as
heumatoid arthritis (RA).
• In this situation, the synovium becomes enlarged and the
cellular composition changes.
• In vitro, RA FLSs exhibit a transformed phenotype
reminiscent of that seen in tumors, whereas the vascular
endothelium displays an increased ratio of apoptotic to
proliferative cells, which is indicative of vascular
remodeling.

7. • Joint inflammation and destruction have been linked to the deregulation of the highly synthetic
fibroblast-like synoviocytes (FLSs), and much of the current understanding of the mechanisms
that underlie synovitis has been collected from studies of FLSs.
• During a proteomic analysis of FLS cells, a novel protein, c19orf10 (chromosome 19 open reading
frame 10),was identified that was produced in significant amounts by these cells.

8. Procedure • Acquiring the cells from RA and OA patients
• Sample preparation
• 2-D gel electrophoresis and in gel digestion
• Mass spectrophotometry
• cDNA cloning and expression constructs
• Antibody production
• Immunofluorescence
• Immunohistochemistry

9. Results • Mass spectrometric analysis of two-dimensional SDS-
PAGE separated synovial fibroblast lysates identified a
16.5-kDa spot as the product of c19orf10.
• The c19orf10 gene is located on chromosome 19p13.3 and
spans approximately 30 kbp.
• A survey of the available cDNA clones suggests the
possibility of three splice variants for c19orf10.The most
common variant,c19orf10.b, has six exons and is
supported by sequence data from 468 clones.
• Domain and pattern searches using InterProScan to query
ProDom, PFAM, SMART, and PRINTS suggest that
c19orf10 does not possess any known domains or motifs.
• A panel of murine monoclonal antibodies was produced
against rhc19orf10 for immunohistochemistry and
immunoassays in an effort to define the distribution of
c19orf10 in synovium.Three hybridomas were selected
based on reactivity with rhc19orf10.

10. (a) The chromosomal localization
of the region containing the
c19orf10 gene is indicated on the
ideogram.
(b) The expanded region of
chromosome 19 containing the
c19orf10 gene.
(c) Alignment of protein products
of c19orf10 splicing variants.

11. The cellular origins of c19orrff10 iinn tthhee ssyynnoovviiuumm. Sections of rheumatoid arthritis
synovial tissue stained with anti-c19orf10 stained red.
(a) Sections stained with anti-CD68 to detect macrophage-like cells stained brown (arrow).
(b) Sections stained with anti-CD55 to detect fibroblasts stained brown (arrows).
There was a clear colocalization with CD55-staining cells, whereas the association with CD63-positive
cells was much less apparent.

12. Conclusion • The protein encoded for by c19orf10 displays novel
structural features.
• The protein is produced by cultured fibroblast-like
synovial cells and cells with fibroblast markers in
synovial tissues.
• The c19orf10 protein is found in significant
concentrations in the synovial fluids of patients with
one of a variety of arthropathies.
• The patterns of synthesis in the synovium may change
with synovial tissue cellularity, possibly indicating that
alterations in production rates or biosynthetic
capabilities may occur in subsets of synovial fibroblasts
in RA and OA.
• However, the role of this protein in synovial biology in
both health and disease remains to be determined.

13. References • The identification and characterization of a novel
protein, c19orf10, in the synovium by Weiler, JohnA.
Arthritis Research andTherapy doi:10.1186/ar2145
• Protein expression facility of the university of
Queensland
• Biologicrop for protein analysis and characterization
• Scientists digitally mimic evolution to create novel
proteins University of North Carolina HealthCare-
science daily.

14. Thank you