Production of Turnip yellow mosaic virus Capsids: The Future in Micronutrient Delivery

1. Production of Turnip yellow mosaic virus Capsids: The Future in Micronutrient Delivery Nicole Dzialowy Dr. Yannis Tzanetakis Dr. Theo Dreher Department of Microbiology

2. Zinc Deficiency • Diets rich in grains and low in protein suffer in zinc • Many developing countries

3. 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

4. Phytate zinc Shortages of Zinc • Lost availability due to interactions with other components of diet • Zn interacts with chelators derived from grains and legumes • Unavailable to be absorbed after binding to chelator Phytate

5. 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

6. 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

7. Zinc Fingers Nanosphere: Turnip yellow mosaic virus Strategy Food-grade Lactobacillus

8. 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

9. 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 Protein Structure of TYMV

10. 5’ 3’ Coatprotein NcoI BamHI 5’ 3’ 5’ 3’ Coatprotein Coatprotein NcoI BamHI Methods 1. Isolate/purify pET 11d 2. Amplify CP RNA with RT-PCR 3. Restriction digest with NcoI and BamHI pET 11d 4. Ligate pET 11d and CP insert pET 11d

11. Methods (cont’d) 5. Transform into DH5αE.coli cells and isolate plasmid 6. Restriction mapping and sequencing CP insert pET 11d DH5α cells 7. Transform into BL21 (DE3)-RP E.coli cells 8. Extract/purify capsid and observe by electron microscope pET 11d BL21 cells T7 RNA Polymerase

12. 1 2 3 4 5 6 bp 5000 1500 1000 750 500 250 1 % TBE agarose gel (Gene Ruler 1kb ladder) Results • Western blot analysis results • Some minor expression, but not as expected Sequence of CP insert

13. 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

14. Acknowledgements • Howard Hughes Medical Institute • Dr. Kevin Ahern • Dr. Yannis Tzanetakis • Dr. Theo Dreher • Dreher Laboratory • Dr. Dave Williams Laboratory