Transcript Production of Turnip yellow mosaic virus Capsids: The Future in

Production of Turnip
yellow mosaic virus
Capsids: The Future in
Micronutrient Delivery
Nicole Dzialowy
Dr. Yannis Tzanetakis
Dr. Theo Dreher
Department of Microbiology
Zinc Deficiency
 Diets rich in grains and low in protein suffer
in zinc
 Many developing countries
Importance of Zinc
 Plays critical role in cell growth and division
 Required for protein and DNA synthesis
 Zinc plays a role in immune system
 Concerns:
 Increased risk of common infections and
growth stunting in children
 Women who are pregnant or lactating
Shortages of Zinc
Phytate
 Lost availability due to
interactions with other
components of diet
 Zn interacts with chelators
derived from grains and
legumes
zinc
 Unavailable to be absorbed
after binding to chelator
Phytate
Zinc Fingers, Solution?
 Protein domains that
are found in a class of
DNA binding proteins
 Cysteine and histidine
residues can bind zinc
 Competitor with
chelators to bind zinc
 Smallest contains 30
amino acids
NH2
COOH
Project Outline
 To develop protein nanospheres as a
protective and nutritious delivery vehicle for
zinc
 Nanosphere = Turnip yellow mosaic virus
 My Summer Project
 To produce TYMV virions in Escherichia coli,
using a recombinant vector that includes the
capsid protein sequence
E.coli
Strategy
Food-grade Lactobacillus
Zinc Fingers
Nanosphere: Turnip yellow mosaic virus
Turnip yellow mosaic virus
(TYMV)
 Background
 Positive strand RNA virus
 Structure
 28 nanometer spherical virion
(capsid)
 Capsid built of 180 copies of the
same protein
 Capsid is stable in the absence of
genome
 Atomic structure is known
Protein Structure of TYMV
 Amino
acid sequence
for coat protein is
known
 Green
and red arrows mark
possible regions for zinc finger
insertion
Both regions face interior of
capsid
Methods
1. Isolate/purify pET 11d
2. Amplify CP RNA with RT-PCR
5’
Coat protein
NcoI
3’
BamHI
3. Restriction digest with NcoI and BamHI
pET 11d
4. Ligate pET 11d and CP insert
5’
3’
5’
Coat protein
pET 11d
NcoI
Coat protein
3’
BamHI
Methods (cont’d)
5. Transform into DH5α E.coli cells
and isolate plasmid
6. Restriction mapping and
sequencing
CP insert
pET 11d
DH5α cells
8. Extract/purify capsid and observe
by electron microscope
7. Transform into BL21 (DE3)-RP E.coli
cells
pET 11d
BL21 cells
T7 RNA Polymerase
Results
1
2
3
4
5
6
bp
5000
1500
1000
750
500
250
1 % TBE agarose gel (Gene Ruler 1kb
ladder)
Sequence of CP insert
 Western blot analysis results
 Some minor expression, but not as expected
Future Work
 Extract/purify capsid, observe on electron
microscope
 Engineer TYMV capsids with zinc fingers in
order to bind zinc
 Explore production of zinc-loaded TYMV
capsid in food-grade Lactobacillus lactis
Acknowledgements
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Howard Hughes Medical Institute
Dr. Kevin Ahern
Dr. Yannis Tzanetakis
Dr. Theo Dreher
Dreher Laboratory
Dr. Dave Williams Laboratory