Download Arginine-glycine-aspartic acid motif incorporated within turnip yellow

ARGININE-GLYCINE-ASPARTIC ACID MOTIFS
INCORPORATED WITHIN THE TURNIP
YELLOW MOSAIC VIRUS COAT PROTEIN
By: Jonathan Halama
Mentors: Dr. Theo Dreher and Josh Powell, MS

Non-enveloped T=3 icosahedral symmetry of identical sequence.

Single stranded positive sense RNA virus.

Coat protein (CP) coded by sub-genomic strand.

180 coat proteins (CP), 60 as 1 of 3 quasi-equivalent sub-units.

60 A sub-units: pentameric capsomeres.

60 B sub-units and 60 C sub-units:
hexameric capsomeres.
RGD MOTIFS AND INTEGRIN


Arginine-glycine-aspartic acid (RGD)
 A tripeptide motif used by Integrins
as an attachment point.
 RGD motifs are found within viral
proteins and facilitate cellular
adhesion.
Integrin (protein receptor)
 Integrin is a ligand used by some cells
and viruses for adhesion and cell
signaling.
 Stem cells up-regulate Integrin
production.
PURPOSE: STEM CELL GROWTH

Utilize RGD motif as a binding site for Integrin
attachment.
 Multiple
binding sites on exterior capsid.
 Regulate stem cell adhesion.
 Control stem cell growth and differentiation.

Mutant TYMV capsids are “vital for designing
compatible biomaterials for tissue engineering
purposes” (Gagandeep Kaur).
HYPOTHESIS
Arginine-Glycine-Aspartic acid (RGD)
motifs can be engineered on the
exterior of Turnip yellow mosaic virus
(TYMV) coat proteins (CP) while
maintaining the capsids: ability to
assemble, structural stability, and
allow binding of Integrin to the RGD
motifs.
COAT PROTEIN MONOMER
RGD 44 Monomer
COAT PROTEIN TRIMER
RGD 44 Trimer
ASSEMBLED CAPSID
ASSEMBLED CAPSID
INTEGRIN/RGD MOTIF INTERACTIONS
INTEGRIN/RGD MOTIF INTERACTIONS
INTEGRIN/RGD MOTIF INTERACTIONS
MUTANTS
Mutants
Subcloning
Agro
infiltrated
Capsid
Extraction
Successful
Assembly
RGD 44
Completed
Completed
Completed
Yes
RGD 55
Completed
Completed
Completed
RGD 61
Completed
Completed
RGD 102
Completed
Completed
RGD 103
Completed
Completed
RGD 161
Completed
Completed
RGD 162
Completed
Completed
RGD 184
Incomplete
Completed
Completed
SUB-CLONING
AGROINFILTRATION AND RECOMBINATION
VIRAL CAPSID EXTRACTION
10X NaPO4
Bentonite solution
RGD TYMV deveined
plant tissue samples
MgSO4
2x Low Speed Spins: 13G
High Speed Spin: 50K RPM
Low Speed Spin: 13G
High Speed Spin: 50K RPM
Resuspend Isolated Capsids
PROTEIN VISUALIZATION
Coomassie gel
Ladder Wild-type RGD 44 RGD 55 RGD 103 RGD 162
27 kDa
15 kDa
Western blot
Western blot prepared using:
•SDS-PAGE.
•Primary antibody:
Rabbit anti-TYMV IgG.
•Secondary antibody:
Goat anti-rabbit IgG Horseradish
Peroxidase (HRP) conjugate.
Coomassie gel prepared using:
•Sodium dodecyl sulfate
polyarcylamide gel electrophoresis
(SDS-PAGE).
•Coomassie Brilliant Dye.
27 kDa
15 kDa
CAPSID VISUALIZATION
RGD 44 (60K)
Electron
Microscopy
RGD 44 (125K)
50 nm
Wild-type
28nm
50 nm
CONCLUSIONS…

RDG 44 expressed the mutant coat protein within the plant
system, and self assembled forming capsids.

RDG 55, 103, or 162 coat proteins were not detected within
the plant system. Future work will determine if the protein was
or was not being expressed.

RDG 61, 62, 102, and 161 require further cloning techniques;
transformation into Argobacterium, argoinfiltrated into the
plant system, and capsid extraction to determine if coat protein
can be expressed and self assemble within plant tissue.
SPECIAL THANKS TO THE FOLLOWING…
HHMI (Howard Hughes Medical Institute)
URISC (Undergraduate Research, Innovation, Scholarship and Creativity)
Dr. Kevin Ahern
The Dreher Lab
Special thanks to Josh Powell
REFERENCES

Kaur, G. / Valarmathi, M.T. / Potts, J.D. / Wang,
Q. , Biomaterials, 29 (30), p.4074-4081,Oct
2008