Lecture 2

1. Lecture 2 • Transgenic Plants • Viral Vectors • Transient Expression

2. Expression in Transgenic Plants Limitations • some crops can not be regenerated • vulnerable to weather • not 100% biologically contained unless via chloroplast transformation Advantages • low initial investment • medium time scale (months) • unlimited scale-up potential • faster than transgenic animals • very low costs <5$/gram • safe, no risk of pathogen contamination • edible crops can be used • active complex proteins (sIgA) 1

3. Transgenic Plants

4. Root and Shoot induction

5. Plant out

6. Plant out/Seeds

7. Tunnels

8. Plant Production: Virus Vectors vs. Transgenic Plants

9. Viral Vectors Viral Vectors for the expression of proteins in plants Yuri Gleba, V. Klimyuk and S. Mariollnnet Current Opinioins in Biotech 2007 18: 134-141

10. Viral Vectors • Plant viral vectors • Clone GOI into virus • Infect plant • Virus replicates • Protein of interest will be produced to high levels

11. Viral vectors - Advantages • Quick (2-3 weeks) • High Yield of protein • Gene is not incorporated • Not heritable trait • Containment of genes

12. First generation Viral vectors • Functionally Viruses • Express all viral genes and GOI • GOI can be fused to CP • GOI expressed from strong promoter • To get GOI into plants • Use DNA or • Viral particles • Rub or spray onto plants • Virus then moves systemically and whole plants gets infected and produces POI

13. Tobacco mosaic virus vector Large Scale Biology Corp. pBSG1057

14. Disadvantages • Only small epitopes are expressed • Low yield (0.04 – 0.3% TSP) • Instability of gene • Loss of trans gene after while • Depends on size of gene – bigger, loss quicker • Limited systemic infection • To increase expression put in linker between CP and GOI

15. Second Generation of Viral Vectors • Use live Agrobacterium as vector • Agrospray or Magnifection • Need to contain recombinant Agro • Make viral genome smaller • Delete all non-essential genes • Ability to infect host • Systemic infection • Replication generally good – keep • Changed viral vectors • Codon usage • Add plant introns

16. First Generation Viral Vectors Second Generation Viral Vectors

17. Viral Vectors • Add T-DNA • Use Agro to deliver proviral replicon • Active replicons in plant cell • Get amplification in each cell and • Movement from cell to cell • Got increase in yield: • g/kg tissue • 50% TSP • Quick 4-10 days

18. First Generation Viral Vectors Second Generation Viral Vectors

19. Infiltration Vacuum – 0.8 bar for 20 sec

20. 6 Days Post-Inoculation 2 Days Post-Inoculation 4 Days Post-Inoculation UV LIGHT NORMAL LIGHT VIRAL VECTOR GFP EXPRESSION

21. Inoculate on plant leaves Harvest in 10 - 14 days Empty Viral Vector Empty Viral Vector Laboratory Work: Few Days Vaccine Gene Vaccine Gene Viral vector with vaccine gene Extract & Purify Time from Gene to Field Production: 6 Weeks to a Few Months Field Production of Vaccines

22. Expression with viral vectors • This method used for any proteins to date: • Human growth hormone – 1mg/kg • Plague antigen • TB antigen • Ab – could not express more than one protein form same viral replicon • Use two viral vectors (heavy and light chain)

23. TMV • ss RNA, 2100 CP • Get very high yield of CP in plants – 10g/kg • Fuse GOI to CP • Can only fuse peptides (20AA) • Linker – bit bigger • Fuse antibody-binding domain of Protein A to CP • Use to purify

24. TMV Protein A fragments Enzymes Ab

25. The ICON deconstructed virus system is the newest of a series of technology advancements B GFP A DSRED

26. Expression Technology Development over time Gene encoding the desired protein. Incorporate into a plant transformation vector for optimized expression in plant cells. Stable expression: Nuclear genome integration. Stable expression: Chloroplast genome integration. Integrate into a viral coding sequence for expression as a “by product” of viral replication. Transient expression: Infect plant to initiate viral replication. Modify viral genome to adapt it into a plant transformation vector for subsequent regulated release as a replicon in transgenic plants. Primarily for antigens/epitopes until very recently Identify protective epitope within antigenic protein. Create viral replicon coding sequence with epitope fusions to coat protein.

27. Transient Expression • Use Agrobacterium infiltration • Plant expression vectors • Advantages • can express more than one gene in same cell • Quick – 3-10 days • High yield • Can produce transgenic plants with same constructs • T-DNA can integrate or remain as episome

28. P35SS 5’UTR GOI (SEKDEL) pA35S SP LB pAnos ColE1 ori Pnos selectable marker bla SAR pTRA RK2 ori P35SS gene of interest RB 5'UT SAR pA35S pTRA Plant Expression Vector • P35SS: CaMV35S promoter withdoubled enhancer • 5’UTR: tobacco etch virus 5’UTR • SP: signal peptide usedfor mammalian expression ofthe antibody chains • SEKDEL: C-terminal ER-retention signal (optional) • pA35S: CaMV35S terminator • SAR: scaffold attachment region

29. Transient Expression via Agrobacterium infiltration of leaves or plants

30. Agrobacterium-infiltration as a useful tool for rapid assessment of gene expression in plants LB RB 35S Gene of Interest OCS [kDa] 97 69 46 wt 945(P) A 945 (A) 1 2 2d 5d HPV L1 Western blot analysis

31. Plant Vaccine Group University of Cape Town direct injection vacuum infiltration Optimising protein production in plants 1. Select optimal constructs using agroinfiltration - simple and rapid method for evaluating protein expression of numerous Agrobacterium constructs 2. Small OR Large-scale infiltration