The document presents research on using a thermostable serine protease from the archaeon Thermococcus kodakarensis to digest abnormal prion protein (PrPSc). The protease, called Tk-subtilisin, is stable in high temperatures, denaturants, and surfactants. Western blot analysis showed that Tk-subtilisin can fully digest PrPSc at 100°C in 3% SDS or industrial surfactants, indicating its potential for decontaminating prion-infected materials. Tk-subtilisin offers advantages over other proteases for prion inactivation due to its thermostability in harsh conditions.

1. Proteolysis of a protease-
resistant, infectious Scrapie
Prion Protein using a Thermal
Stable Serine Protease
Presented by:
Alexander Ward
Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis
KOD1
Authors: Yuichi Koga & Shun-ichi Tanaka & Akikazu Sakudo & Minoru Tobiume & Mutsuo Aranishi & Azumi Hirata
& Kazufumi Takano & Kazuyoshi Ikuta & Shigenori Kanaya

2. Discovery of Prion Disease (PrPSc)
 Neurodegenerative, progressive,
fatal
 Unknown origin
 No isolated bacteria or viruses?
 Spongiform neural tissues
 Types –
 Infectious
 Variant
 Genetic
"Pathology & Laboratory Medicine." Tulane University. Tulane University School of Medicine, n.d. Web.

3. Infectious PrPSC
Diaz-Espinoza, Rodrigo, and Claudio Soto. "High-resolution Structure of Infectious Prion Protein: The Final Frontier." Nat
Struct Mol Biol Nature Structural & Molecular Biology 19.4 (2012): 370-77. Web.

4. Types of Prion Diseases (PrPSc)
Prusiner, Stanley B., Michael R. Scott, Stephen J. DeArmond, and Fred E. Cohen. "Prion Protein Biology." Cell, Volume 93,
Issue 3, 1 May 1998. Web.
Disease Host Mechanism of Pathogenesis
Kuru Fore people Infection through ritualistic
cannibalism
varient Creutzfeldt-Jakob
Disease
Humans Infection from bovine prions?
Scrapie Sheep Infection in genetically
susceptible sheep
Bovine Spongiform
Encephalopathy
Cattle Infection with prion-
contaminated Meat and Bone
Meal
Chronic Wasting Disease Mule deer, elk Unknown
Feline Spongiform
Encephalopathy
Cats Infection with prion-
contaminated beef

5. What is a Prion Protein? (PrPC)
 Membrane protein
 Found in mammalian brain,
spinal and eye tissue
 3 CTD α-helices, 2 linked by a
disulfide bond
 Unstructured NTD
 NTD has a high affinity for
divalent cations
Baral, P. K., M. Swayampakula, and M.N.G. James. "4MA7." RCSB PDB. PubMed, 22 Jan. 2014.
Diaz-Espinoza, Rodrigo, and Claudio Soto. "High-resolution Structure of Infectious Prion Protein: The Final Frontier." Nat
Struct Mol Biol Nature Structural & Molecular Biology 19.4 (2012): 370-77. Web.

6. What is a Prion Protein? (PrPC)
 Associated with
 Immunoregulation
 Signal transduction
 Copper binding
 Synaptic transmission
 Induction of apoptosis
 Protection against apoptotic
stimuli
Baral, P. K., M. Swayampakula, and M.N.G. James. "4MA7." RCSB PDB. PubMed, 22 Jan. 2014.
Diaz-Espinoza, Rodrigo, and Claudio Soto. "High-resolution Structure of Infectious Prion Protein: The Final Frontier." Nat
Struct Mol Biol Nature Structural & Molecular Biology 19.4 (2012): 370-77. Web.

7. What’s the difference between PrPC and
PrPSc?
Diaz-Espinoza, Rodrigo, and Claudio Soto. "High-resolution Structure of Infectious Prion Protein: The Final Frontier." Nat
Struct Mol Biol Nature Structural & Molecular Biology 19.4 (2012): 370-77. Web.
a – β-helical model
b – β-spiral model
c – β-sheet model
Amyloid state doesn’t equal
neurodegeneration

8. Decontamination Procedure - WHO
 Incineration
 Autoclave/chemical methods
for dry goods
 Autoclave/chemical methods
for heat-resistant instruments
 Chemical methods for surfaces
or heat sensitive instruments
"WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. Report of a WHO Consultation, Geneva,
Switzerland, 23-26 March 1999." WHO. N.p., Mar. 1999.

9. Organism – Thermococcus kodakarensis KOD1
(Tk)
 Hyperthermophilic Archaea
 Temp. 60-100o C (opt. 85o C)
 pH 5-9 (opt. 6.5)
 Salt conc. 1-5% (opt. 3%)
 Strictly anaerobic
"Thermococcus Kodakaraensis Genome Project." Imanaka Laboratory,
Graduate School of Engineering, Kyoto University, Japan, 17 Feb.
2005. Web. 11 Nov. 2015.

10. Tk-subtilisin
Tanaka, S., H. Matsumura, Y. Koga, K. Takano, and S. Kanaya. "3A3P." RCSB PDB. PubMed, 22 Oct. 2010. Web. 10 Nov.
2015.
 Stable in high temperature,
denaturants, and surfactants
 7 binding sites for Ca2+
 Subtilisin is a serine protease

11. Serine Proteases
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.

12. Serine Proteases: Hydrolytic Mechanism
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.
 His 57 acts as a general base,
deprotonating Ser 195
 Ser 195 nucleophilic attack the
carbonyl C of the polypeptide
backbone

13. Serine Proteases: Hydrolytic Mechanism
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.
 Oxyanion collapses back to form a
double bond between the original
carbonyl C
 His 57 acts as a general acid to
protonate the amine leaving group

14. Serine Proteases: Hydrolytic Mechanism
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.
 H2O regenerates the active site
 How to fit the substrate in the
active site?

15. Denaturation of Proteins
 Protonation or
deprotonation
 Anion or cation interaction
Ophardt, Charles E. "Denaturation Protein." Denaturation Protein. Elmhurst Chemistry Department, 2003. Web. 10 Nov.
2015.

16. Denaturation of Proteins
 Interruption through
stronger hydrogen
bonding molecules
Ophardt, Charles E. "Denaturation Protein." Denaturation Protein. Elmhurst Chemistry Department, 2003. Web. 10 Nov.
2015.

17. Denaturation of Proteins
 Reduction of disulfide
bonds
Ophardt, Charles E. "Denaturation Protein." Denaturation Protein. Elmhurst Chemistry Department, 2003. Web. 10 Nov.
2015.

18. Denaturation of Proteins
 Detergents
 Interact with non-polar amino acid
residues
 Small polar molecules
 Depends on structure and amino
acid composition
 Heat can also denature proteins
Ophardt, Charles E. "Denaturation Protein." Denaturation Protein. Elmhurst Chemistry Department, 2003. Web. 10 Nov.
2015.

19. Methods - Enzyme Activity Assay
 Azocasein –
 Measures enzymatic activity using azocasein as a substrate
 Consists of casein conjugated to an azo-dye
 N-succinyl-AAPF-p-nitroanilide (Sigma)
 Used to test enzyme activity in protein denaturing conditions
 Cleavage of 4-nitroanilide yields 4-nitroaniline, yellow under basic conditions
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2
(2015): 2113-20.

20. Results - Stability of Tk-subtilisin against
Denaturants
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2
(2015): 2113-20.
SDS 8M Urea 6M GdnHCl
● - Tk-subtilisin
∆ - proteinase K
□ - Subtilisin Carlsberg

21. Results - Impact of Surfactants on Tk-
subtilisin
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2
(2015): 2113-20.
0.1%
1%

22. Methods - Western Blot
"Western Blot." PPT PowerPoint Drawing Diagrams, Templates, Images, Slides. Motifolio, n.d. Web. 11 Nov. 2015.

23. Western Blot Analysis
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2
(2015): 2113-20.
 1 and 1’ – PrPSc
 2 and 2’ – PrPSc and
Proteinase K
 3 and 3’ – PrPSc and Tk-
subtilisin
 4 and 4’ – PrPSc, Tk-subtilisin
and Proteinase K

24. Western Blot Analysis
 1 and 1’ – PrPSc
 2 and 2’ – PrPSc and DFP
 3 and 3’ – PrPSc and Tk-
subtilisin
 4 and 4’ – PrPSc, Tk-
subtilisin and DFP
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2
(2015): 2113-20.

25. Western Blot Analysis
 A concentration of
0.002mg/mL is
enough to digest
60µg of PrPSc in the
sample in 3% SDS,
at 100o C for 10
minutes.
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2
(2015): 2113-20.

26. Western Blot Analysis
 Two non-ionic industrial
surfactants, Emulgen PP290
and 147
 Emulsifiers
 Incubated at 100oC for 10
minutes
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2
(2015): 2113-20.

27. Western Blot Analysis
 sCJD brain
homogenate
 Incubated at 100oC
for 10 minutes
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2
(2015): 2113-20.

28. Conclusion
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical Journal 108.2
(2015): 2113-20.

29. Special Thanks
 Dr. Mary Karpen
 GVSU Chemistry Department
 Authors of the article

30. Questions

31. Additional Information
 DFP – Diisopropyl fluorophosphate – Inhibits some proteases. (C6H14FO3P)
 Promazine – Phenothiazine, a class of antipsychotics

32. DFP to Inactivate Serine Proteases
Jakubowski, Henry. "Chymotrypsin." Chymotrypsin. UC Davis, 20 Nov. 2013. Web. 10 Nov. 2015.

33. Issues with Decontamination Procedures
- WHO
 It is recommended to discard instruments used in surgery of brain, spinal cord
or eyes of patients with a family history of PrPSc.
 The size of the macerate and thermostability of the PrPSc, increasing porous
load autoclaving temperature or time is not a reliable decontamination
standard.
 It has been reported that PrPSc agents are more resistant to inactivation of
autoclaving when infected brain-tissue becomes dried onto glass or metal
surfaces.
 Macerated samples were more resistant to porous load autoclaving compared
to intact tissues.
Taylor, D.m. "Inactivation of Prions by Physical and Chemical Means."Journal of Hospital Infection 43 (1999): S69-S76.

34. Table of Industrial Surfactants used in
Tk-subtilisin activity measurements
Koga, Yuichi, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya. "Proteolysis of Abnormal Prion Protein with a
Thermostable Protease from a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1." Biophysical
Journal 108.2 (2015): 2113-20.

35. Decontamination Procedure - WHO
"WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. Report of a WHO Consultation, Geneva,
Switzerland, 23-26 March 1999." WHO. N.p., Mar. 1999.
Chemical disinfectants
variably effective
Gaseous disinfectants
ineffective
Physical processes
variably effective
Urea (6M) Ethylene Oxide Autoclaving at 121oC for
15 minutes
SDS (5%) Formaldehyde Boiling in 3% SDS
Alcohol Ionizing UV or
microwave radiation