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MODIFIED BACTERIOPHAGE
LAMBDA PROCAPSIDS FOR USE AS A DRUG
DELIVERY PLATFORM.Presentation by:Nicole Rowley and Kyle Koshland
◎ Advantages of using viruses
◎ What are Procapsids?
◎ Optimization of Procapsid Production
◎ Procapsid Modification
◎ Future Directions
Presentation Overview
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
◎ Procapsid without DNA or tail◎ Bacteriophage of E. coli
What Are Lambda Procapsids?
http://pdbj.org/emnavi/emnavi_detail.php?id=1507
2 Main Goals:
1. Procapsid Prep
2. Procapsid Modification
2 Main Goals:
1. Procapsid Prep
2. Procapsid ModificationFor drug
packaging
For quantification of modification
sites
2 Main Goals:
1. Procapsid Prep
2. Procapsid Modification
And the difficulties of finding a procedure that works!
1.Lambda Procapsid Prep
http://pdbj.org/emnavi/emnavi_detail.php?id=1507
Plasmid TransformationDNA 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
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
Ladd
er
+Con
trol
Resea
rch P
rep
SDS Gel Shows No ProductkDa
260
160
11080
60 50
40
3020
Capsid Protein
Confirmation of Product
Ladd
er
+ Con
trol
Resea
rch P
rep
Capsid Protein
kDa260
160
110 80
60
50 40
30
20 1510
Ladder
Research Prep
kDa260
160
110
80
60
5040
30
20
Capsid Protein
Intact Procapsid Confirmed
Intact
Denatured
2 Main Goals:
1. Procapsid Prep
2. Procapsid Modification
2 Main Goals:
1. Procapsid Prep
2. Procapsid Modification
2.Procapsid Shell Modifications
https://www.carthage.edu/live/files/723-heidifenskemaxwell-maxhurickposterpdf
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
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
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
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
Further Experimentatio
n◎ Procapsid modifications
○ Attach linker and targeting protein
◎ Mouse studies
○ Inflammation in lupus mouse model
○ Blood-Brain barrier
Any Questions?
Thank You
FPLC: Procapsid (Denatured)
FPLC Results
