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. 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 zinc Phytate

5. Zinc Fingers, Solution? COOH NH 2  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

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

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. 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

10. Methods 1. Isolate/purify pET 11d2. Amplify CP RNA with RT-PCR 3. Restriction digest with NcoI and BamHI 4. Ligate pET 11d and CP insert BamHI NcoI 5’3’ Coat protein BamHI NcoI 5’3’ pET 11d Coat protein 5’3’

11. Methods (cont’d) DH5α E.coli cells 5. Transform into DH5α E.coli cells and isolate plasmid 6. Restriction mapping and sequencing 7. Transform into BL21 (DE3)-RP E.coli cells 8. Extract/purify capsid and observe by electron microscope pET 11d CP insert DH5α cells pET 11d BL21 cellsT7 RNA Polymerase

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