Production of Production of Turnip Turnip yellow mosaic virusyellow mosaic virus Capsids: The Future Capsids: The Future

in Micronutrient in Micronutrient DeliveryDelivery Nicole DzialowyNicole Dzialowy

Dr. Yannis Tzanetakis Dr. Yannis Tzanetakis Dr. Theo DreherDr. Theo Dreher

Department of MicrobiologyDepartment of Microbiology

Zinc DeficiencyZinc Deficiency Diets rich in grains and low in protein suffer Diets rich in grains and low in protein suffer

in zinc in zinc Many developing countriesMany developing countries

Importance of ZincImportance of Zinc Plays critical role in cell growth and Plays critical role in cell growth and

divisiondivision Required for protein and DNA synthesisRequired for protein and DNA synthesis

Zinc plays a role in immune systemZinc plays a role in immune system Concerns:Concerns:

Increased risk of common infections and Increased risk of common infections and growth stunting in childrengrowth stunting in children

Women who are pregnant or lactatingWomen who are pregnant or lactating

Shortages of ZincShortages of Zinc Lost availability due to Lost availability due to

interactions with other interactions with other components of dietcomponents of diet

Zn interacts with chelators Zn interacts with chelators derived from grains and derived from grains and legumeslegumes

Unavailable to be absorbed Unavailable to be absorbed after binding to chelatorafter binding to chelator

Phytate

zinc

Phytate

Zinc Fingers, Solution?Zinc Fingers, Solution?

COOH

NH2

Protein domains that Protein domains that are found in a class of are found in a class of DNA binding proteinsDNA binding proteins

Cysteine and histidine Cysteine and histidine residues can bind zincresidues can bind zinc

Competitor with Competitor with chelators to bind zincchelators to bind zinc

Smallest contains 30 Smallest contains 30 amino acids amino acids

Project OutlineProject Outline To develop protein nanospheres as a To develop protein nanospheres as a

protective and nutritious delivery vehicle for protective and nutritious delivery vehicle for zinczinc Nanosphere = Nanosphere = Turnip yellow mosaic virusTurnip yellow mosaic virus

My Summer ProjectMy Summer Project To produce TYMV virions in To produce TYMV virions in Escherichia coliEscherichia coli, ,

using a recombinant vector that includes the using a recombinant vector that includes the capsid protein sequencecapsid protein sequence

E.coli

StrategyStrategyFood-grade Lactobacillus

Nanosphere: Turnip yellow mosaic virus

Zinc Fingers

Turnip yellow mosaic Turnip yellow mosaic virusvirus (TYMV) (TYMV) BackgroundBackground

Positive strand RNA virusPositive strand RNA virus StructureStructure

28 nanometer spherical virion 28 nanometer spherical virion (capsid)(capsid)

Capsid built of 180 copies of the Capsid built of 180 copies of the same proteinsame protein

Capsid is stable in the absence of Capsid is stable in the absence of genomegenome

Atomic structure is knownAtomic structure is known

Protein Structure of Protein Structure of TYMVTYMV

Amino acid sequence Amino acid sequence for coat protein is for coat protein is knownknown

Green and red arrows mark Green and red arrows mark possible regions for zinc finger possible regions for zinc finger insertioninsertion

Both regions face interior of Both regions face interior of capsidcapsid

Methods Methods 1. Isolate/purify pET 11d 2. Amplify CP RNA with RT-PCR

3. Restriction digest with NcoI and BamHI

4. Ligate pET 11d and CP insert

BamHINcoI5’ 3’

Coat protein

Coat protein

BamHINcoI

5’ 3’

pET 11d

pET 11d

Coat protein5’ 3’

Methods (cont’d)Methods (cont’d)5. Transform into DH5DH5αα E.coli E.coli cells cells

and isolate plasmid6. Restriction mapping and

sequencing

7. Transform into BL21 (DE3)-RP E.coli cells8. Extract/purify capsid and observe

by electron microscope

pET 11d

CP insert

DH5DH5αα cells cells

pET 11d

BL21 cells T7 RNA Polymerase

ResultsResults

Western blot analysis resultsWestern blot analysis results Some minor expression, but not as expectedSome minor expression, but not as expected

1 % TBE agarose gel (Gene Ruler 1kb ladder)

1 2 3 4 5 6

bp

250

500

7501000

1500

5000

Sequence of CP insert

Future WorkFuture Work Extract/purify capsid, observe on electron Extract/purify capsid, observe on electron

microscopemicroscope Engineer TYMV capsids with zinc fingers in Engineer TYMV capsids with zinc fingers in

order to bind zincorder to bind zinc Explore production of zinc-loaded TYMV Explore production of zinc-loaded TYMV

capsid in food-grade capsid in food-grade Lactobacillus lactisLactobacillus lactis

AcknowledgementsAcknowledgements Howard Hughes Medical InstituteHoward Hughes Medical Institute Dr. Kevin AhernDr. Kevin Ahern Dr. Yannis TzanetakisDr. Yannis Tzanetakis Dr. Theo DreherDr. Theo Dreher Dreher LaboratoryDreher Laboratory Dr. Dave Williams LaboratoryDr. Dave Williams Laboratory