1. Role of Human Kallikrein 7
Protease in Metastasis
Interns:
Shay Seguin
Sharmila Mitra
Nick Wong
Nick Higdon
Mentor: Jennifer Guerrero
Advisor: Professor Patrick Daugherty
SIMS, University of California, Santa Barbara

2. 90% of Cancer Deaths Are Associated With
the Spreading of a Tumor
Tumor
Blood Vessel
Secondary
Tumor
http://www.wpclipart.com/

3. Role of Protease in Biological Functions
Cell Adhesion
Proteins
Dead Skin Cell
Healthy Skin Cell
hK7 Protease
hK7

4. Role of Protease in the Spreading of Cancer
Cancer Cells
Healthy Cells
Cell Adhesion
Substrates
Protease
Courtesy of Jennifer Guerrero

5. Which Substrates Break Down the Fastest
hK7 Protein
Substrate
Substrates
• Fibronectin
• Desmocollin-1
• Desmoglein-1
• Desmoglein-2
• E-cadherin
Binding
Cleavage

6. Determine if an Inhibitor Can Effectively Block hK7
hK7
Protein
Substrate
Inhibitor
No Binding
No Cleavage

7. Using FRET Protein to Test Protease Activity
Yellow: no cleavage
Cyan: cleavage
Substrate
FRET: Förster Resonance Energy Transfer
Courtesy of Jennifer Guerrero
hK7

8. Preparing the Bacteria for Protein Production
Plasmid DNA
Gene Coding
FRET Substrate
Arabinose Promoter
Courtesy of Jennifer Guerrero openwetware.org
E. coli
Target Protein

9. 0.00
0.10
0.20
0.30
0.40
0.50
0 30 60 90 125
Time (minutes)
Add Arabinose
“On Switch”
Culturing E. coli for Substrate Production
Cell Density
(1x10^9 cell/mL)

10. Add BPER II Detergent
Spin Down in Centrifuge
Purifying FRET Proteins in E. coli
Target Protein
Excess Protein
Dead E. coli
Live E. coli

11. Target Protein
Excess Protein
Nickel Beads
Mesh Filter
Purifying FRET Proteins in E. coli
250 mM Imidazole Wash

12. Measuring Substrate Cleavage With
Fluorimeter
hK7 FRET
Substrate
Substrates
• Fibronectin
• Desmocollin-1
• Desmoglein-1
• Desmoglein-2
• E-cadherin
+

13. 0%
20%
40%
60%
80%
100%
0 40 80 120 160 200 240
PercentageofSubstrateCleaved
Time (Minutes)
Ratio of Substrate to Substrate Cleavage

14. Percentage of Substrate Cleaved in 1 Hour
10%
77%
81%
92%
95%
Desmoglein-2
Fibronectin
E-cadherin
Desmocollin-1
Desmoglein-1

15. Demonstrating the Effect of an Inhibitor
on hK7 Activity
3%
95%
With Inhibitor
Without Inhibitor
Percentage of Substrate Cleaved in 1 Hour
Desmoglein-1

16. Cancer Cells
Healthy Cells
Cell Adhesion
Substrates
Protease With Inhibitor
Protease Inhibitors as Cancer Therapeutics

17. Acknowledgements
Special thanks to:
Mentor: Jennifer Guerrero
Advisor: Professor Patrick Daugherty
SIMS Staff:
Ofelia Aguirre
Jonathan Cook
Stephanie Mendes
Dean Morales
Andrew Dawson
Kori Lay
Kuang Wei
Mary Lou Bailey