This master's thesis project examines serine proteases and their role in immunity. The student, Luke Morton, conducted experiments on chicken cathepsin G (CTSG) and Chinese alligator mast cell protease-1 (MCP-1) including recombinantly expressing the proteases in HEK 293 cells, quantifying the proteases, activating them to mimic natural cleavage, testing substrate specificity using phage display and chromogenic substrates, and comparing the proteases' activity levels against PBS controls over multiple days. The conclusions suggest the proteases have highly specific cleavage specificity addressed by upstream/downstream interactions or multiple substrate binding sites. Future work could involve augmenting the phage display library or testing different substrates and membrane interactions.

1. Serine Proteases, A Basis of
Immunity Through Evolution
Master Thesis Project
By Luke Morton
Lars Hellman Lab

2. Serine Proteases? Prolific
Throughout Biological systems

3. Granulocytes

4. Granulocyte Proteases

5. Granulocyte Proteases

6. Protease Specificity By Family
The S1 Pocket of the
protease conveys an aspect
of the cleavage specificity,
also dictating the P1
position nomenclature.

7. MrBayes MCMC (Markov Chain
Monte Carlo) Bioinformatic
association map based on sequence
homology and surrounding genes,
indicating likely protease specificities
from various species.

8. Loci for Chicken Cathepsin G &
Chinese Alligator Mast Cell
Protease-1
“[Serine Proteases] evolved in parallel with the immune cells in which they are
now expressed; thus, […] variations open a window onto the origins and
development of mammalian immune cells in their current state of complexity
and specialization.” Caughey 2006

9. Quantification of Insert & Vector

10. Transfection into HEK 293
Major changes from previous pCEP4 vector:
• Introduction of puromycin resistance
versus the previous hygromycin.
• Signal sequence from human BM40
• EcoRI sites upstream of the ampicilin
resistance gene moved after extension of a
polylinker to also include a NotI site
EBNA-1: Ebstein-Barr virus nuclear antigen 1,
responsible for keeping cell in an altered state
utilizing viral proteins to take advantage of cellular
machinery (SV40).
Col E1: Copy number regulation system designed
to reduce plasmid expression collapse.
P CMV: Cytomeglovirus promoter
Ori P: origin of replication.

11. Expression & Harvesting
•Once the vector has been selected for in
the HEK 293 cells, they are grown and
expanded, producing variable quantities of
protein into the media, which is then
collected and purified using Ni-NTA beads.
•SDS-PAGE shows the qualitative
concentrations of the elutions done with
imidazole.

12. Quantification of Protease
Chicken
CTSG High Elution
Dilution
Factor X-values (OD) Y-Values (eq) Y-valuesxDF Avg Conc.
5x 0,04 0,1095 0,5475
2x 0,092 0,2703 0,5406 0,5ug/ul
1x 0,153 0,5459 0,5459
Low Elution
5x 0,019 0,0534 0,267
2x 0,046 0,1261 0,2522 0,2ug/ul
1x 0,07 0,1968 0,1968
Alligator MCP-1
5x 0,025 0,0699 0,3495
2x 0,06 0,1663 0,3326 0,3ug/ul
1x 0,072 0,203 0,203
Comparative quantification of the protease
concentration is done through:
• Bradford Standard Assay
•Bovine Serum Albumin Dilution Assay
(eq) y=50.724x3-1.8662x2+2.6431x+0.0035

13. Activation of CTSG & MCP-1
EFHHHHHDDDDKIVGGRRARPHAWPFMVSL
QLRGGHFCGSTLIAPNFVMSAAHCCVANVNV
RAVRVVLGAHNLSRREPTRQV…
•Imitates zymogen cleavage of proteases in
natural system.
•Found also in compliment activation,
fibrinogen and kinin systems.
•Removes small protein flap from active site ,
allowing interaction.

14. Recombinant substrate assays
V2:VVRRAAAG
V3:VVRRRAAG
HC: VVLFSEVL
OC: VGLWLDRV
HCV6: VVLLSEVL
CF1: RVTGMSLV

15. Chromogenic Substrate Assay
• Results most likely from Enterokinase
•Prefix (Suc, Boc, Z, Ac) to make
substrate soluble in H2O
•Suffix (pNA-paranitroaniline)

16. Phage Display
Chicken CTSG
Chinese Alligator MCP-1

17. Phage Display Results
0.00
5.00
10.00
15.00
20.00
25.00
30.00
1 2 3 4 5
Ratiovs.PBS
Day
CTSG & MCP-1 vs. PBS 29/3/16
CTSG
MCP-1
PBS
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
1 2 3 4 5
Ratiovs.PBS
Day
CTSG & MCP-1 vs. PBS 18/4/16
CTSG
MCP-1
PBS
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
1 2 3 4 5 6
Ratiovs.PBS
Day
CTSG & MCP-1 vs. PBS 02/05/16
CTSG
MCP-1
PBS
•Chicken CTSG & Alligator MCP-1 PFU
ratios versus PBS.
•PFU was calculated as an average
from usually 2-3 different dilution sets
for each day.
•See a selection arc but towards the
end of the week it crashes, otherwise it
would continue.

18. Conclusions
• Chicken CTSG & alligator MCP-1 have a extremely specific
cleavage specificity that is possibly addressed further up or
downstream from the P5-P5’ site or with multiple interaction sites
with its substrates.
• Many proteases interact with proteoglycans in and outside of
granules and on the cell membrane, without these specific
configurations or charge alignments there could be reduced affinity
to primary targets.
• Augmenting phage display library (or trying different
recombinant/chromogenic substrates) for better substrate
representation, or attempt to have a protease/substrate/membrane
interaction etc.
• Future…

19. Acknowledgements
• A big thanks to…
Lars Hellman
Srinivas Akula
Zhirong Fu
Gurdeep Chahal
Payal Banerjee
The A8:2 Corridor!