My thesis evalution slide

1. Cloning, Expression, Purification and
Enzymological Characterization of
NS2B/NS3 Protease / RNA Helicase protein of
Japanese Encephalitis Virus.
Chakard Chalayut
Advisor: Asst. Prof. Gerd Katzenmeier, Ph.D.
Laboratory of Molecular Virology
Institute of Molecular Biology & Genetics

2. Japanese Encephalitis Virus
-Flaviviridae family
-Mosquito-borne neurotropic
flavivirus
•causes severe central
nerve system diseases

3. Japanese Encephalitis Virus
Culex tritaeniorhynchus.
Source : vietnammedicalpractice.com
Source: fehd.gov.hk

4. Japanese Encephalitis Virus
JEV causes severe
central nerve system
diseases such as
50,000 Cases
10,000
p o l i o mye l i t i s - l i ke
30% fatality rate
acute flaccid paralysis,
aseptic meningitis and
encephalitis
Source:wonder.cdc.gov
Source: cdc.gov

5. Prevention and treatment of
JEV disease
Drug No drug exist
Vaccine development Available vaccine
Elimination of mosquitoes
Mosquitoes control breeding places

6. Molecular biology of Japanese Encephalitis Virus
Source : molecular-virology.uni-hd.de

7. The NS2B
• 130 aa Hypothetical model
• activating domain NS2B-NS3 complex
central hydrophilic region
(Falgout et al, 1993)
• 3 membrane spanning parts
hydrophobicity plot
ฺBrinkworth et al, 1999
51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95

8. The NS3
Protease
NTPase
•Chymotrypsin-like fold
2-β barrel Helicase
RNA domains
•Inactive alone
Theoretical model from PDB
•Enzyme’s pocket is small
2I84

9. The NS3 protease
Complexation with NS2B cofactor
•
conformational change serine protease
NS3
alteration of the enzyme pocket
additional substrate binding 20 kDa
domain site
•catalytic residues His51,
Asp75, Ser135

10. Background
• NS2B(H) JE - NS3p Den did not cleaved Den
polyprotein but NS2B(H) Den - NS3p JE cleaved JEV
polyprotein.
• The C-terminal portion of Den NS2B is required for
interaction with Den NS3 to activate protease.
• Jan. L R et al, 1995

11. Background
• Ser to Ile60 were essential region required for NS3
46
protease activity.
• Ala substition of Trp
50, Glu55, and Arg56
in NS2B
shown significantly reduced NS3 protease activity.
• Lin. C W et al,2007

12. Objective
• to perform cloning of the NS2B-NS3 portion of the JEV
polyprotein, express in E.coli and biochemically purify
to determinants of clevage activity and cofactor
requirement will be analyzed and compared to dengue
virus.
• The second objective is to study differences in
substrate specificity and inhibitors by using peptide
substrates incorporated with fluorogenic or
chromogenic reported groups.

13. Method & Result
pLS with NS2B-NS3 JEV
NS2B(H) NS3p
SOE-PCR
NS2B(H)-NS3p

14. NS2B(H)
NS2B NS3p NS3
NS2B(H) NS3p

15. - Control
NS2B(H)
NS2B(H)
NS3 protease
NS3 protease
NS3 protease
- Control
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Figure 1 : The PCR product NS2B(H) JEV amplified Figure 2 : The PCR product NS3protease JEV
from NS2B-NS3 JEV (Lane 3 and 4). The size of amplified from NS2B-NS3 JEV (Lane 3 to 5 ). The size
NS2B(H) was 187 bp. of NS3 protease was 594 bp.

16. NS2B(H)-NS3p
- Control
NS2B(H)-NS3p
- Control
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Figure 3 : The SOE-PCR product NS2B(H)- Figure 4 : The NS2B(H)-NS3protease JEV
NS3protease JEV (Lane 2 to 5 ). The size of (Lane 3 ) after digested with BamHI and KpnI.
NS2B(H)-NS3 protease was 765 bp. The size of NS2B(H)-NS3 protease was 765 bp.

17. Method & Result
pTrcHis Den with NS2B-NS3 Den
NS2B(H) NS3p
SOE-PCR
NS2B(H)-NS3p Den

18. NS3 protease
NS3 protease
- Control
- Control
NS2B(H)
NS2B(H)
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1000 900
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800 700
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Figure5 : The PCR product NS2B(H) Den amplified Figure 6 : The PCR product NS3protease Den amplified
from pTrc NS2B-NS3 Den (Lane 2 and 3). The size of from pTrc NS2B-NS3 Den (Lane 3 and 4 ). The size of
NS2B(H) was 187 bp. NS3 protease was 594 bp.

19. NS2B(H)-NS3p
NS2B(H)-NS3p
NS2B(H)-NS3p
NS2B(H)-NS3p
NS2B(H)-NS3p
NS2B(H)-NS3p
NS2B(H)-NS3p
- Control
- Control
1500
1000
900
1500 800
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Figure 7 : The SOE-PCR product NS2B(H)-NS3protease Figure 8 :The NS2B(H)-NS3protease Den (Lane 3 and
Den (Lane 2 and 6). The size of NS2B(H)-NS3 protease 4 ) after digested with BamHI and KpnI. The size of
was 765 bp. NS2B(H)-NS3 protease was 765 bp.

20. Method & Result
pTrcHis A
Digest with BamHI Digest with KpnI
Digest with KpnI Digest with BamHI
Gel Electrophoresis & Clean with Gel Extraction Kits

21. with BamHI
Digested pTrcHis A
with KpnI
Digested pTrcHis A
pTrcHis A
23.13 kb
9.42 kb 23.13 kb
6.56 kb
9.42 kb
4.56 kb 6.56 kb
4.56 kb
2.32 kb
2.03 kb
2.32 kb
2.03 kb
Figure 9 : The pTrcHis A in lane 2 without digestion Figure 10 : The pTrcHis A in lane digested with
BamHI and KpnI in lane 3 and 4.In line 1 is
pTrcHis A without digestion.

22. Method & Result
NS2B(H)-NS3p JEV
pTrcHis A
NS2B(H)-NS3p Den
Ligation & Transformation
Site Screening & Digest with Restriction Enzyme

23. Dengue Clone 1-5
Dengue Clone 11-15
Dengue Clone 16-20
Dengue Clone 21-25
Dengue Clone 6-10
Dengue Clone 4
Dengue Clone 5
Dengue Clone 6
Dengue Clone 8
Dengue Clone 9
Dengue Clone 1
Dengue Clone 2
Dengue Clone 3
Dengue Clone 7
NS2B(H)-NS3p
JEV Clone 1
JEV Clone 1
pTrcHis A
23.13 kb
23.13 kb
9.42 kb
9.42 kb 6.56 kb
6.56 kb
4.56 kb
4.56 kb
2.32 kb 2.32 kb
2.03 kb 2.03 kb
700 bp
Figure 12 : The pTrcHis NS2B(H)-NS3protease JEV
Figure 11 : The pTrcHis NS2B(H)-NS3protease JEV
(lane 12) and pTrcHis NS2B(H)-NS3protease Den
(lane 8) and pTrcHis NS2B(H)-NS3protease Den (lane
clone 1-9 (lane 3 to 11) was digested with BamHI and
3 to 7) was digested with BamHI.
KpnI.

24. Conclusion
• The NS2B(H)-NS3p JEV and NS2B(H)-NS3p
Den was successfully PCR and ligated into
pTrcHis A plasmid.
• The digestion of the recombinant vector
shown the expect band of pTrcHis with the
insert, then it need to purify and sequence
the plasmid to make sure,that is the correct
plasmid.

25. Future Work
• Check another clone to get more positive
clone.
• Construct NS2B(H)Den-NS3p JE and
NS2B(H) JE-NS3p Den.
• Retransform into E.coli C41.
• Purification of Protein.
• Enzyme assay.

26. Thank you for your
attention.