1. MODIFIED
BACTERIOPHAGE LAMBDA
PROCAPSIDS FOR USE AS
A DRUG DELIVERY
PLATFORM.
Presentation by:
Nicole Rowley and Kyle Koshland

2. ◎ Advantages of using viruses
◎ What are Procapsids?
◎ Optimization of Procapsid Production
◎ Procapsid Modification
◎ Future Directions
Presentation Overview

3. Advantages of Viruses
◎ Variety of shapes and sizes in
nanometer size range
http://epidemiologiamolecular.com/wp-content/uploads/2011/08/clip_image0064.jpg
◎ Non-toxic
◎ Inexpensive to produce
◎ Amino acid residues can be
chemically and genetically
modified

4. ◎ Procapsid without DNA or tail
◎ Bacteriophage of E. coli
What Are Lambda Procapsids?
http://pdbj.org/emnavi/emnavi_detail.php?id=1507

5. 2 Main Goals:
1. Procapsid
Prep
2. Procapsid
Modification

6. 2 Main Goals:
1. Procapsid
Prep
2. Procapsid
ModificationFor drug
packaging
For
quantification
of modification
sites

7. 2 Main Goals:
1. Procapsid
Prep
2. Procapsid
Modification

8. And the difficulties of
finding a procedure that
works!
1.
Lambda Procapsid
Prep
http://pdbj.org/emnavi/emnavi_detail.php?id=1507

9. Plasmid Transformation
DNA GpE/GpD Plasmid
Origin of
Replication
Ampicillin
Resistance
GpE/GpD
T7 Promotor
Operator
http://vignette2.wikia.nocookie.net/mmg-233-2013-genetics-genomics/images/e/e2/Transformation.png/revision/latest?cb=20131002205536
Heat
Shock
LacI

10. Procapsid Formation
http://viralzone.expasy.org/all_by_protein/3945.html
◎ Scaffolding proteins assist
○ Bonds break up completion
○ Not occurring in λ procapsid
◎ Assemblies by Brownian Motion

11. SDS Gel Shows No Product
kDa
260
160
110
80
60
50
40
30
20
Capsid
Protein

12. Confirmation of Product
Capsid
Protein
kDa
260
160
110
80
60
50
40
30
20
15
10
kDa
260
160
110
80
60
50
40
30
20
Capsid
Protein

13. Intact Procapsid Confirmed
Intact
Denatured

14. 2 Main Goals:
1. Procapsid
Prep
2. Procapsid
Modification

15. 2 Main Goals:
1. Procapsid
Prep
2. Procapsid
Modification

16. 2.
Procapsid Shell
Modifications
https://www.carthage.edu/live/files/723-heidifenskemaxwell-maxhurickposterpdf

17. Maleimide Modification
http://www.drugtargetreview.com/4442/content-type/industry-news/genticel-announces-positive-preclinical-proof-of-concept-results-for-its-multivalent-hpv-therapeutic-vaccine-gtl002/
R = Cy5
Intact
Denatured
Dye
Intact

18. NHS Ester Modification
http://www.drugtargetreview.com/4442/content-type/industry-news/genticel-announces-positive-preclinical-proof-of-concept-results-for-its-multivalent-hpv-therapeutic-vaccine-gtl002/
R = Cy5
Intact Intact

19. Quantification labels
◎ Used Beer’s Law
○ Extinction coefficient
◎ Average successful labeled amino acids
○ Lysines:
◉ 274 Cy5/Intact Procapsid
○ Cysteines:
◉ 208 Cy5/Intact Procapsid
http://www.pharmaceuticalonline.com/doc/lambert-beer-law-0001

20. Quantification stability
◎ Stability after conjugation
○ Lysine chemistry:
◉ 92.75% Intact
○ Cysteine chemistry:
◉ 50.67% Intact
◎ Calculated from area under curve
○ Ratio of intact to broken procapsids

21. Further
Experimentation
◎ Procapsid modifications
○ Attach linker and targeting protein
◎ Mouse studies
○ Inflammation in lupus mouse
model
○ Blood-Brain barrier

22. Any Questions?

23. Thank You

25. FPLC: Procapsid (Denatured)
FPLC Results